Method for detecting target nucleic acid sequence using cleaved complementary tag fragment and a composition therefor

ABSTRACT

The present invention relates to a method and a composition for detecting a target nucleic acid sequence using a cleaved complementary tag fragment. Specifically, the present invention relates to a method for linking a complementary tag sequence to a PCR primer so that a tagging can be produced by a restriction enzyme during a PCR reaction, diversifying the complementary tag sequence to be linked to each primer by utilizing factors such as length and nucleic acid combination, etc., and distinguishing the target sequence using the same. According to the present invention, a cleaved complementary tag fragment (CCTF) under stringent conditions is a complementary sequence to any sequence at the 5′ end linked to the primer and cannot be formed unless a PCR reaction and a restriction enzyme reaction occur, and the cleaved single strand is formed only when hybridization to the target sequence occurs and a primer extension product complementary to the target sequence is formed, so as to have a higher degree of accuracy secured by reading the cleaved single strand. In addition, the CCTF can be used to identify a plurality of target nucleic acid sequences by selecting various analytical techniques and analysis equipment according to a user&#39;s intention. For example, a result can be confirmed rapidly and accurately in genetic testing, identification of organisms in a sample, diagnosis of microbial or viral infection, etc.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of the U.S. application Ser. No. 16/095,695, filed Oct. 23, 2018, the disclosure of which is incorporated herein in its entirety by reference.

TECHNICAL FIELD

The present invention relates to a method for detecting target nucleic acid sequence using cleaved complementary Tag fragment and a composition therefor, and specifically relates to a method of identifying the amplified product by ligating a template of tag capable of producing a marked substance to a primer that specifically reacts with the target sequence, thereby synthesizing and releasing the tag by restriction enzyme activity in the PCR reaction and introducing it into the reaction solution. Also, the present invention relates to a method for generating and identifying a tag characterized in that the tag generated during a reaction using only one kind of template sequence of a tag for one target sequence is analyzed in various analyzing apparatuses to identify the tag, and a composition used in the method.

BACKGROUND ART

Polymerase Chain Reaction (PCR) is one of techniques very usefully utilized in detecting and analyzing low concentration nucleic acids. The detection of the nucleic acid is based on the complementarity of the double strand oligonucleotide sequences and the extension reaction of each DNA polymerase, and the target nucleic acid sequence can be detected using this (Barry et al., Current Opinion in Biotechnology, 12; 21, 2001).

Multiple PCR is a method that can simultaneously amplify nucleic acids of multiple target sequences, and is relatively fast and simple compared to other methods, and thus plays a very large role in diagnosis field such as genetic test, identification of organisms in samples, and microbial or viral infection, etc.

The most common method for confirming the results of such multiplex PCR is to design primers by varying an amplification product size of the target sequence as desired in PCR, and to analyze the size of the amplified product by electrophoresis of the PCR result, and then to confirm as to whether amplification of the target sequence is made. In this case, the number of genes that can be amplified at one time is limited to 3 to 4 experimentally because there is a restriction that the size of the amplification product should be limited within a narrow range, due to that the efficiency of amplification depends on the size of the amplification product that can be generated during the PCR reaction and thus a uniform amplification efficiency cannot be guaranteed. In this case, it also occurs the case that the size of the desired gene amplification product may overlap. Therefore, there is a limit to the interpretation of the detection method when the multiple PCR is analyzed depending on the size.

Real-time PCR guarantees a confirmation of a rapid PCR result in confirming the PCR results, and it can identify as to whether the amplification is made by marking fluorescent material regardless of the size of the amplified product. The methods performing and detecting Real-time PCR can be divided into intercalating method and probe method, wherein the intercalating method is referred to a method of confirming fluorescence intensity by inserting fluorescent substance between double-stranded base sequences. Since this method cannot distinguish the amplification products forming the double strands, and can observe all of them as the fluorescence of the same wavelength. Therefore, it has a limit on identifying the amplification product by each target sequence to detect and identify at least one amplified product simultaneously. The probe method is a method of detecting the amplified product by reading the fluorescence value of the probe designated for each target sequence and, in the case of using this method, since the amplification product can be detected only in the number of analyzable fluorescence channels of a device to be used, the multiple analysis over the number of fluorescent channels is not suitable for this.

Therefore, studies were continuously carried out to insert the tag during PCR to enable the maximum number of multiple analysis.

In the case of Luminex's xTAG technology, a constant base sequence comprised of a random array of thymine (T), adenine (A), and guanine (G), which constitutes the nucleic acid, was set and named xTAG. It is a method comprising inserting xTAG sequence into the primer to be located the xTAG sequence at the end in the amplification of the target sequence to be observed, so that the xTAG was inserted into the amplification product during the PCR procedure, and secondarily joining the xTAG with a bead to which the complementary sequence to xTAG attached to form a complementary bond between the two base sequences, detecting the target using the same, and analyzing the target sequence with fluorescence of the bead. In this method, even though the xTAG does not participate in the amplification, if the primer is not completely removed after the amplification, it has problems that there is a possibility that it binds to the complementary xTAG of the bead to recognize the mark, and an error occurs that the complementary sequence of xTAG forms non-specific reaction by PCR and thus non-specific target is detected (U.S. Pat. Nos. 7,645,868 and 8,624,014).

In order to solve this problem, studies has been continuously performed that a tag is constructed during the PCR reaction, the tag does not affect the PCR reaction, the maximum numbers of multiple detections are possible.

DISCLOSURE Technical Problem

The present invention is derived to solve the above problems and to meet the above needs and the object of the present invention is to provide a method for solving the uncertainty which can be occurred when the results are determined depending on the length of the generated product in amplifying and analyzing a target sequence using an amplification reaction such as PCR, and for solving the restriction to the maximum numbers of amplification that can be identified in multiple detection.

The another object of the present invention to provide a method for improving accuracy by solving errors due to non-specific amplification which can be caused by the use of artificial sequence as a tag itself in identifying a target sequence amplification by forming the tag.

Technical Solution

In order to accomplish the above object, the present invention provides a primer with the structure comprising a target sequence and a non-complementary random nucleic acid sequence and sequentially comprising a restriction enzyme recognition sequence and a nucleic acid sequence complementary to the target sequence.

In one embodiment of the present invention, the restriction enzyme recognition sequence is preferably one selected from the group consisting of Pho I, PspGI, BstNI, TfiI, ApeKI, TspMI, BstBI, BstEII, BstNI, BstUI, BssKI, BstYI, TaqI, MwoI, TseI, Tsp45I, Tsp509I, TspRI, Tth111I, Nb.BsmI, Nb.BsrDI, Nt.BspQI, Nt.BstNBI restriction enzymes and Nick restriction enzymes, but is not limited thereto.

In another embodiment of the present invention, the said primer is preferably one that a modified dNTP inserted at the cleavage site of the restriction enzyme recognition sequence of the primer, for the purpose of that a cleaved by-product other than the cleaved complementary tag fragment allow not to participate in the reaction, and the modified dNTP to be inserted into the cleavage site is phosphorothioated dNTP, dNTP containing 7-deazapurine, or a 2′-O-methyl nucleotide (2′-OmeN) in a DNA template, but is not limited thereto.

In another embodiment of the present invention, it is preferable, but not limited, that the primer is from 5 mers or more to 50 mers or less in length of the cleaved complementary tag fragment as generated.

In one embodiment of the present invention, the primer is one or more one selected from the group consisting of SEQ ID NOS: 1, 3, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 74, 76, 78, 80, 82, 86, 115, 117, 119, 121, 123, 125, 127, 129, 131, 151, 153, 155, 156, 159, 161, 164, 166, 168, 170, 204, 205, 207, 218, 220 and 222, but is not limited thereto.

Furthermore, the present invention provides a method for forming a tag to be used in classifying and analyzing the kinds of the target sequences amplified in the Polymerase Chain Reaction, and identifying it, comprising:

-   -   a) hybridizing a target sequence with a primer of the present         invention comprising a template of a tag for generating the tag         that is a cleaved complementary tag fragment,     -   b) generating the complementary tag fragment cleaved from the         primer by the activity of a restriction enzyme when the         amplification procedure is proceeded by the hybridization of a)         and introducing it into a reaction solution, and     -   c) identifying the generated cleaved complementary tag fragment         through an analyzer to confirm the presence of the target         sequence.

In one embodiment of the present invention, it is preferable to analyze the mass of the cleaved complementary tag fragment to identify the cleaved complementary tag fragment in the above method, and the instrument used for the mass spectrometry is preferably a matrix-assisted laser desorption-ionization-time-of-flight mass spectrometer ((MALDI-TOF MS), a Liquid Chromatography Mass Spectrometer, or a High Performance Liquid Chromatography Mass Spectrometer, but is not limited thereto.

In another embodiment of the present invention, the mass per unit electric charge (m/z) of the cleaved tag fragment to be used for mass spectrometry is preferably from greater than 0 to 10000 Da or less, but is not limited thereto.

In another embodiment of the present invention, in order to preserve the mass of a cleaved complementary tag fragment to be used in mass analysis during the amplification process, it is preferable to use DNA polymerase that the function of adenine-addition elongation effect (A tailing) at the 3′end, which is an intrinsic property of the nucleic acid polymerase, is inhibited, but is not limited thereto.

In another embodiment of the present invention, it is preferable to analyze the fluorescence signal using the oligonucleotide that is tagged by fluorescence and Quencher and has the complementary sequence of the cleaved complementary tag fragment as the identification method of the cleaved complementary tag fragment, but it is not limited thereto.

In another embodiment of the invention, it is preferable to analyze the dissociation temperature and melting peak by varying the inherent dissociation temperature at which the double strand of the oligonucleotide and the cleaved complementary tag fragment are dissociated into a single strand, and to identify the presence of the target sequence by identifying the cleaved complementary tag fragment in the method, but is not limited thereto.

In yet another embodiment of the present invention, the oligonucleotide is preferably 5 or more in length, but is not limited thereto.

In another embodiment of the present invention, it is preferable to attach a quencher to the nucleotide at the 3′end of the oligonucleotide in order to prevent elongation of the base sequence from the oligonucleotide in the method, but is not limited thereto.

In another embodiment of the present invention, it is preferable to identify the complementary tag fragment cleaved by analyzing the cycle threshold (C) value of the fluorescence signal of the oligonucleotide, but not limited thereto.

In a preferred embodiment of the present invention, it is preferable to identify causative organisms of a sexually transmitted disease in the said method, and the sexually transmitted disease causative organism is preferable one selected from the group consisting of Chlamydia trachomatis, Neisseria. Gonorrhea, Mycoplasma hominis, Mycoplasma genitalium, Trichononas vaginalis, Ureaplasma urealyticum, Ureaplasma parvum, Candida albicans, Gardnerella vaginalis, Herpes simplex virus 1, Herpes simplex virus 2, Treponema pallidum, but is not limited thereto.

The present invention also provides a composition for diagnosing sexually-transmitted diseases, comprising the primer of the present invention as an effective component.

In another embodiment of the present invention, it is preferable to identify the causative organism of gastrointestinal tract disease, wherein the causative organism of gastrointestinal tract disease is selected from the group consisting of Rotavirus A, Astrovirus, Adenovirus F40, Adenovirus F41, Norovirus GI and Norovirus GII, but is not limited thereto.

The present invention also provides a composition for diagnosing a gastrointestinal disease agent comprising the primer of the present invention as an effective component.

In another preferred embodiment of the present invention, it is preferable to identify a human papilloma virus in the method, and the subpopulations of the human papilloma virus is preferably selected from the group consisting of types 16, 18, 33, 35, 51, 53, 59, 68a, and 82, but is not limited thereto.

The present invention also provides a composition for diagnosing HPV comprising the primer of the present invention as an effective component.

In another preferred embodiment of the present invention, it is preferable to identify a causative organism of the respiratory disease in the method, and the causative organism of the respiratory disease is one being selected from the group consisting of Influenza A/H1N1, Influenza A/H3N2, influenza A/H1N1/2009pdm, influenza B, Parainfluenza 1, Parainfluenza 3, Respiratory syncytial virus A, Respiratory syncytial virus B, Human metapneumovirus, Adenovirus, but is not limited thereto

The present invention also provides a composition for the diagnosis of respiratory diseases, comprising the primer of the present invention as an effective component.

In another preferred embodiment of the present invention, the method is preferably a single nucleotide polymorphism (SNP), wherein the single base mutation is preferably one selected from the group consisting of r6265 of the Brain-derived neurotrophic factor gene (BDNF gene), but is not limited thereto.

The present invention also provides a composition for analyzing the BDNF gene rs6265 gene comprising the primer of the present invention as an effective component.

Hereinafter, the present invention will be described.

The present inventors have tried our best to develop the method that can perform a multiplex amplification reaction on a large number of targets at one time by clearly distinguishing each amplification product through an easier, faster and more efficient method in preforming the amplification reaction and can analyze the results.

As a result, so as to be able to generate a nucleic acid sequence which can be used as a tag in an amplification reaction, when a sequence serving as a template for a tag was inserted into a primer, and only tag was cleaved by a restriction enzyme, we confirmed that the generated tag can play a role as the tag for detecting the target sequence and also identified that it can identify the amplification efficiently and rapidly than other existing methods in the multiplex amplification reaction analysis by applying it to various analysis methods, and thus, has been completed the present invention.

The present invention relates to a method of forming tags to be used for sorting and analyzing kinds of amplified target sequences during a PCR reaction.

In particular, the present invention is characterized in comprising the steps of: (1) hybridizing a target sequence with a primer comprising a template of a tag for generating the tag, (2) generating the tag from the template of the tag using a restriction enzyme during the PCR reaction, and (3) analyzing the generated tag with various analysis equipment to identify the tag.

(1) As the step for hybridizing a target sequence with a primer (CTPO-Cleavable Tag Primer Oligonucleotide, hereinafter referred to as CTPO) comprising a template of a tag for generating the tag (CCTF-Cleaved Complementary Tag Fragment, hereinafter referred to as CCTF); wherein CTPO comprises a sequence non-complementary to the target sequence (the template of CCTF), followed by a restriction enzyme recognition sequence and a nucleic acid sequence complementary to the target sequence, and the nucleic acid sequence site complementary to the target sequence located at the 3′end hybridizes with the target sequence, thereby playing a role as a primer during the PCR reaction,

(2) as a step for generating and releasing CCTF from CTPO by the activity of a restriction enzyme in the amplification process; wherein the restriction enzyme recognition sequence is inserted into the amplified product elongated from the above-described CTPO, and CCTF is generated by the activity of the thermostable restriction enzyme recognizing it and introduced into the reaction solution,

(3) as the step for analyzing and identifying the generated CCTF through various analysis equipment to confirm existence of a target nucleic acid sequence; wherein the mass of the generated CCTF is measured to identify the type of CCTF, and the amplified product is sorted to confirm the presence of the target nucleic acid sequence, or the fluorescence is emitted during the procedure that the oligonucleotide composed of the sequence complementary to the generated CCTF and tagged with the fluorescence and the quencher (Signal Capture Oligonucleotide—SCO, hereinafter referred to as SCO) and CCTF are hybridized to form a double strand and dissociate again into a single strand, and such inherent dissociation temperature is analyzed to identify the type of CCTF, and to identify whether the amplification of the target nucleic acid sequence is occurred or not.

Hereinafter, the present invention will be described in detail.

In step (1), prior to hybridizing the CTPO and the target sequence, the structure of CTPO is divided into a template portion of the CCTF, a restriction enzyme recognition sequence, and a sequence complementary to the target as shown in the following Formula 1.

5′-A-B-C-3′  Formula 1

The A site in the structural formula 1 is comprised of a random sequence to be a template of the CCTF, and the complementary sequence of the CCTF template, that is, the CCTF site, is elongated by amplifying it after annealing with the target sequence and then the CCTF site is released by the restriction enzyme during the amplification. The released CCTF is characterized by being a random sequence having 5 or more oligonucleotides in length so that it can be specifically analyzed as a tag. Random sequences can be used in any sequence that does not create a by-product during the PCR reaction. The nucleotide sequence to be used as a template for CCTF is free from any sequence that does not cause a hybridization reaction during the amplification reaction

B is a restriction enzyme recognition sequence, which means a specific recognition sequence of restriction enzymes and Nick restriction enzymes having thermal stability that can be used during amplification. For example, it includes Pho I, PspGI, BstNI, TfiI, ApeKI, TspMI, BstBI, BstEII, BstNI, BstUI, BssKI, BstYI, TaqI, MwoI, TseI, Tsp45I, Tsp509T, TspRI, Tth111I, Nb.BsmI, Nb.BsrDI, Nt.BspQI, Nt.BstNBI, etc.

Most preferably, among them, PspGI can be used, and the restriction enzyme used in Example of the present invention is PspGI.

The modified dNTP is inserted into a site cleaved by the restriction enzyme in the restriction enzyme recognition sequence of CTPO so as not to exist and participate the cleaved by-products other than CCTF in the reaction. Examples thereof include phosphorothioated dNTPs, dNTPs containing 7-deazapurine, or 2′-O-methyl nucleotides (2′-OMeN) in DNA templates, etc. The prior art, PNAS 89 (1992) 392-396 and Nucleic Acids Research 20 (1) 199155-61 can be applied to the present invention. Most preferably, a phosphothiolated bond is inserted into the cleavage site among the recognition sequence to prevent the cleavage of the template of CCTF by a restriction enzyme, thereby securing a template capable of generating CCTF and to prevent a by-product which can be generated by releasing the template of CCTF into the reaction solution, thereby increasing the efficiency of the reaction. It represents the effects of the invention different from the prior art, SDA (Strand Displacement Amplification) method (US Pat. No. 92-819,358) in view of that it generates CCTF and prevents the template to inflow to the reaction solution.

The C site shown in the structural formula 1 means a part after the restriction enzyme recognition sequence up to the 3′end, and is composed of a target specific sequence so that it binds specifically to the target during amplification so as to maintain its role as a primer.

In step (2), when the amplification product is formed by CTPO, and the amplified product present in the double strand is cleaved to CCTF by the restriction enzyme and released into the reaction solution, the appropriate concentration of the restriction enzyme to be used can be varied depending on the purpose of use. In addition, the results are different depending on the type of polymerase to be used, which can be also varied depending on the purpose of use. For example, when CCTF is formed for the purpose of mass spectrometry, it is preferable that the weight of CCTF should be kept constant regardless of the amplification process and should not reflect the intrinsic property of the nucleic acid polymerase. Therefore, a nucleic acid polymerase having no adenine addition extension effect (A tailing) at the 3′end, which is an intrinsic property of the nucleic acid polymerase, should be selected and used. Among the nucleic acid polymerase enzymes that do not make A tailing, Phusion polymerase, Vent polymerase, Deep Vent polymerase, Bst polymerase, etc. are present.

However, when CCTF analysis method using other techniques than mass analysis is applied, there is no variation in the results due to the A tailing effect, and thus, any polymerase can be used.

In order to increase the efficiency of the restriction enzyme to generate CCTF and to maximize the effect by promoting the influx into the reaction solution, a restriction enzyme reaction time can be further added during the PCR process. Reaction time, reaction temperature, etc. can be applied differently depending on the kind of the specific restriction enzyme and the reaction intention.

In step (3), as the step that the generated CCTF is analyzed through various analysis equipment to identify the target nucleic acid sequence, when the mass of the generated CCTF is directly analyzed, the kinds of CCTF are diversified through recombination of length and sequence, Mass spectrometry such as MALDI-TOF MS, LC MS and HPLC MS can be used to observe the intrinsic mass of the generated CCTF, and the amplified target sequence can be identified and identified using the said mass. It is preferable to observe it through MALDI-TOF MS, the range of mass of CCTF which is easy to observe is 1200 Da or more. The amplification products can be observed by forming various CCTFs in the mass range as above.

The amplified target sequence can be identified by observing the fluorescence signal of CCTF, and this is the method which comprises hybridizing CCTF with SCO which is tagged with the fluorescence and the quencher so that the generated CCTF can provide the fluorescence signal at the inherent dissociation temperature, and is the sequence complementary to the CCTF having the inherent dissociation temperature, analyzing the fluorescence signal at the inherent dissociation temperature, and confirming the generation of CCTF, thereby identifying the presence of the target nucleic acid sequence.

For the release of CCTF, as described above, the use concentration of the restriction enzyme is designated according to the purpose of use, and the kind of the polymerase is not related to the A tailing unlike the mass analysis. The CCTF released from the amplification product and introduced into the reaction solution reacts with the SCO present in the reaction solution, wherein the component of the SCO is as follows.

The complementary sequence of CCTF exists to enable hybridization with CCTF from the 5′end to the 3′ end and the sequence of SCO is determined by CCTF length, sequence recombination depending on CCTF. In order to diversify the kinds of tags in step (1), the combination of the length and the sequence may be designed differently to give the inherent dissociation temperature of CCTF and SCO, such as in the case using the method such the length of CCTF and the method of sequence recombination, etc. In this case, the SCO is composed of a complementary sequence of CCTF, and the fluorescent substance is contained in the sequence, and the position of the fluorescent substance is possible in anywhere at least a certain length apart from the quencher. At the 3′end of the SCO, a blocker is positioned so that SCO serves as a primer during the reaction to prevent the nucleotide sequence from elongation. Spacer C3, Phosphat, ddC, Inverted END and Quencher, etc. may be used as the blocker, but not limited thereto. In particular, when the quencher is located at the 3′end of SCO, the SCO is served as a primer during the reaction to prevent the nucleotide sequence from elongation, and simultaneously hybridizes with CCTF to suppress the emission of the fluorescent material by the FRET phenomenon, before it forms a double strand with CCTF. By using a quencher in combination with a substance preventing nucleotide sequence elongation, an unnecessary modification reaction can be shortened in the production of SCO, thereby increasing the yield of the production reaction and further reducing the manufacturing cost. By using the hybridization of CCTF generated during the reaction with SCO contained in the reaction, it can be identified as to whether CCTF is generated by identifying the dissociation of the double strand with the fluorescence and analyzing it to confirm whether CCTF is generated due to the target sequence, and then the target sequence can be identified. The range of temperature that can be defined by the inherent dissociation temperature of the SCO is ˜95° C., and if there is no interference of the dissociation temperature of each double strand, there is no limitation in defining the inherent dissociation temperature for each fluorescent substance.

The combination of SCO's fluorophore and quencher can be exemplified as Alexa Fluor 350, Alexa Fluor 405. Alexa Fluor 430. Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATO Rho6G, ATTO 540Q, ATTO 550. ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATO Thio12, ATO 580Q, ATTO Rho101, ATO 590, ATTO Rho13, ATTO 594, ATTO 610, ATTO 612Q, ATTO 620, ATTO Rho14, ATO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATO 725, ATTO 740, ATTO MB2, AMCA, AMCA-S, BODIPY FL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY TR, BODIPY 630/650, BODIPY 650/665, Biosearch Blue, CAL Fluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red 610, CAL Fluor Red 635, Pulsar 650, Quasar 570, Quasar 670, Quasar 705. FAM, Fluorescein, Fluorescein-C3, Calcein, Carboxyrhodamine 6G, Carboxy-X-rhodamine (ROX), Cascade Blue, Cascade Yellow, Cy2, Cy3, Cy5, Cy3.5, Cy5.5, Cy7, Dansyl, Dapoxyl, Dialkylaminocoumarin, 4′,5′-Dichloro-2′,7′-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin, HEX, Hydroxycoumarin, IRD40, IRD 700, IRD 800, JOE, Lissamine rhodamine B, LC Red 610, LC Red 640, Marina Blue, Methoxycoumarin, Naphthofluorescein, NED, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, PyMPO, Pyrene, Phycoerythrin, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodol Green, 2′,4′,5′,7′-Tetra-bromosulfonefluorescein, Tetramethyl-rhodamine (TMR), Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X. TET, VIC, Yakima Yellow, BMN-Q460, DDQ-1, Dabcyl, BMN-Q530, BMN-Q535, Eclipse, Iowa Black FQ, BHQ-1, TQ2, IQ4, QSY-7, BHQ-2, TQ3, DDQ-II, BBQ-650, Iowa Black RQ, QSY-21, BHQ-3, etc., and may also include any fluorescent material and quencher.

In addition, the reaction between SCO and CCTF occurs simultaneously with the amplification reaction and the CCTF formation reaction, and in this case, by utilizing the fact that the double strand formation ratio of SCO represents a similar efficiency to the amplification amount of the target sequence, the Ct graph of the SCO having the inherent dissociation temperature can be made, and by using this, it is possible to identify the target sequence in a different manner from the inherent dissociation temperature analysis method.

The above content made by the solving means of the present invention will be described in more detail as the most preferable embodiment through the Examples of the present invention.

Advantageous Effects

In the method of the present invention, since an arbitrary tag (CCTF) is generated and cleaved by restriction enzymes during the amplification reaction, the double strand of the restriction enzyme recognition sequence is not formed before the amplification reaction of the artificial sequence (CTPO) added to form the tag and thus, there is no possibility that it is randomly cleaved; since the tags are generated only by the reaction products specifically generated to the target sequence during PCR, the method of the present invention has the high accuracy for forming CCTF, and can obtain more delicate analysis results than the existing PCR result analysis depending on the length of the PCR amplification product or the specificity of the specific sequence; and the method of the present invention can distinguish and interpret amplification products specifically even if various kinds of amplification products are produced in the same length. In addition, since the analysis of the resultant CCTF can be applied to most of the analysis using base sequence, the device for interpretation can be selected and applied ordinarily. In particular, the method of the present invention can be used in the fields of diagnosis, etc., which require rapid multiple analysis using an amplification reaction.

DESCRIPTION OF DRAWINGS

FIG. 1 is a representative diagram illustrating the formation process of CTPO and CCTF used in a PCR reaction, and an example for the analysis of CCTF, as a schematic diagram of CCTF formation.

FIG. 2 shows the results of the formation of CCTF and MALDI analysis in dual target PCR. CTPO was designed to form different CCTFs for each target sequence, amplified, and analyzed by MALDI, and as a result, a peak corresponding to the masses of CCTF 1 obtained by amplifying Neisseria gonorrhoeae (NG) and cleaving it and CCTF2 obtained by amplifying Mycoplasma hominis (MH) and cleaving it, were observed.

FIG. 3 shows the results of Real-time PCR Melting Peak analysis for causative organisms of sexually transmitted diseases. As the results representing the multiple target dissociation temperature measurements to each target of Chlamydia trachomatis(CT), Neisseria gonorrhea (NG) Mycoplasma hominis(MH), Mycoplasma genitalium(MG), Trichomonas vaginalis(TV), Ureaplasma urealyticum(UU), Ureaplasma parvum(UP), Candida albicans(CA), Gardnerella vaginalis(GV), Herpes simplex virus 1 (HSV 1), Herpes simplex virus 2(HSV 2), Treponema pallidum(TP) and Internal Control (IC), the peak was observed at the inherent dissociation temperature that each SCO has (CT:FAM 80° C., NG:HEX 76.5° C., MH:HEX 68° C., MG:CaRed610 67.5° C., TV:Quasar670 71.5° C., UU:CalRed610 77° C., UP:FAM 77° C., CA:FAM 65° C., GV:Quasar670 78.5° C., HSV 1: Quasar705 73.5° C., HSV 2:Quasar705 79° C., TP:Quasar705 66° C., IC:Quasar670 63.5° C.) (a)(b)(c)(d)(e)(f), and no peak of SCO that visualized CCTF was observed when the target sequence was not added in the same composition (g).

FIG. 4 shows the results of Real-time PCR Melting Peak analysis for the causative organism of the gastrointestinal diseases. As the results representing the multiple inherent dissociation temperature measurements to each target of Rotavirus A(RVA), Astrovirus(AstV), Adenovirus F40(AdV 40), Adenovirus F41(AdV 41), Norovirus GI(NoV GI), Norovirus GII(NoV GII) and External Control, the peak was observed at the inherent dissociation temperature that each SCO has (RVA:HEX 78° C., AstV:CalRed610 78° C., AdV 40:CaRed610 67° C., AdV 41:CaRed610 67° C., NoV GI:FAM 68° C., NoV GII:FAM 84° C., EC:HEX 69° C.) (a)(b)(c)(d), and no peak of SCO that visualizes CCTF was observed when the target sequence was not added in the same composition (e).

FIG. 5 shows the results of Real-time PCR Melting Peak analysis for Human Papilloma Virus (HPV) detection. As a result of multiple inherent dissociation temperature measurements of each target of type 16, type 18, type 33, type 35, type 51, type 53, type 59, type 68a, type 82 and IC, the peak was observed at the inherent dissociation temperature that each SCO has (type 16: HEX 76.5° C., type 18: FAM 78° C., type 33: Quasar670 71° C., type 35: Quasar670 71° C., type 51: Quasar670 71° C., type 53: Quasar670 71° C., type 59: Quasar670 71VC, type 68a: Quasar670 71° C., type 82: Quasar670 71° C., IC: Quasar670 67.5° C.) (a)(b)(c)(d), and no peak of SCO that visualizes CCTF was observed when the target sequence was not added in the same composition (e).

FIG. 6 shows the result of Real-time PCR Melting Peak analysis for detection of respiratory disease-induced virus. As a result of multiple inherent dissociation temperature measurements of each target of Influenza A/H1N1(H1), Influenza A/H3N2(H3), Influenza A/H1N1/2009pdm (2009pdm), Influenza B (Flu B), Parainfluenza 1 (PIV 1), Parainfluenza 3 (PIV 3), Respiratory syncytial virus A (RSV A), Respiratory syncytial virus B (RSV B), Human metapneumovirus (MPV), Adenovirus (AdV), External control (EC), the peak was observed at the inherent dissociation temperature that each SCO has (H1: FAM 67.5° C., H3: FAM 76.5° C., 2009pdm: FAM 86.5° C., Flu B: CalRed610 83.5° C., PIV 1: Quasar670 66° C., PIV 3: Quasar670 74° C., RSV A: HEX 63.5° C., RSV B: CalRed610 72° C., MPV: HEX 86° C., ADV: Quasar670 85° C., EC: CalRed610 68.5t) (a)(b)(c)(d)(e), and no peak of SCO that visualizes CCTF was observed when the target sequence was not added in the same composition (f).

FIG. 7 shows the results of Real-time PCR Melting Peak analysis to analyze the genotype of rs6265, a single nucleotide polymorphism of BDNF gene. As a result representing the multiple inherent dissociation temperature measurements of each target of mutant A/A, wild type G/G and heterozygote A/G, the peak was observed at the inherent dissociation temperature that each SCO has (A/A: 76.5° C., A/G: 76.5° C.

75° C., G/G 75° C., IC: 66° C.) (a)(b)(c)(d), and no peak of SCO that visualizes CCTF was observed when the target sequence was not added in the same composition (e).

FIG. 8 shows the results of real-time PCR Ct graph. As a result representing fluorescent amplification curves and standard curves of SCO under the experimental condition of a multi-real-time polymerization chain reaction experiment of Neisseria gonorrhea (NG), Mycoplasma. hominis (MH), Ureaplasma. parvum (UP) in which genomic DNA of each of the above causative organism was diluted by 10-folds from 100 pg/ul concentration, (a) graph shows the results of fluorescence amplification curves plotted when three target sequences are present at each concentration simultaneously, (b) graph shows a negative result plotted when all three target sequences are not included. When the standard curve is represented by a single fluorescence amplification curve of the graph corresponding NG of (a) graphs, it can be represented as (c) and (d), and the graph corresponding to MG graph can be represented as (e) and (f), and the curve corresponding to UP can be represented as (g) and (h).

MODE FOR INVENTION

Hereinafter, the present invention will be described in detail with reference to Examples. These examples are for illustrative purposes only and thus, are not interpreted to limit the scope of the present invention.

Example 1. Formation of CCTF and MALDI Analysis in Dual Target PCR

This experiment was conducted to prove that the CCTF formed during the PCR reaction for the detection of multiple target sequences can be detected in a target-specific manner by analyzing the mass using MALDI-TOF MS. In Example 1, the causative organism of sexually transmitted diseases, DNAs of Neisseria. gonorrhoeae (NG) and Mycoplasma. Hominis (MH) were used as the targets.

1. Target Template DNA, and Primers Manufactured by Sequence Specific Manner

The forward primers of NG and MH targeting in this example were manufactured based on the method described in the Detailed Description of the Invention as CTPO. The 5′end of the forward primer was an arbitrary nucleotide sequence consisting of a sequence non-complementary to the DNA of NG and MH so that it could be used as a template of CCTF, and a restriction enzyme recognition sequence was consecutively located thereon. The sequence after the restriction enzyme recognition sequence up to the 3′end is composed of a sequence complementary to the target region of the DNA of NG and MH, and plays a role as a primer. In addition, the 5′end of forward primer is composed of a different number of nucleotides with each other and has a different mass value for each CCTF generated, in order to design that the amplification products can be distinguished from each other as the mass when CCTF is formed. The reverse primer is consisted of a sequence complementary to the target site of the DNA of NG and MH.

Primer information and target sequence information being amplified and generated are as follows.

Primer 1: (SEQ ID. NO: 1) 5′-TGAACTAT* 

TCCGACGTTTCGGTTGTGTTGAAACACCGCCCGG-3′ Primer 2: (SEQ ID. NO: 2) 5′-GCTCCTTATTCGGTTTGACCGG-3′ Primer 3: (SEQ ID. NO: 3) 5′-ATCTATGATA* 

TTTAGCTCCTATTGCCAACGTATTGG-3′ Primer 4: (SEQ ID. NO: 4) 5′-TGTGTGGAGCATCTTGTAATCTTTGGTC-3′

Amplified product 1: GenBank: CP012028.1/Position (start-end): 251416-251506

(SEQ ID. NO: 5) 5′-TGAACTAT* 

 TCCGACGTTTCGGTTGTGTTGAAACACCGCC CGGAACCCGATATAATCCGCCCTTCAACATCAGTGAAAATCTTTTTTTTA ACCGGTCAAACCGAATAAGGAGC-3′

Amplified product 2: GenBank: AJ243692.1/Position (start-end): 835-944

(SEQ ID NO: 6) 5′ ATCTATGATA* 

 TTTAGCTCCTATTGCCAACGTATTGGAAA AAAACTTTGGTATTGAAAAAGGATT TATGACAACAGTCCACTCATATACAGCAGACCAAAGATTACAAGATGCTC CACACA-3′

The bold and slanted font of the Primer sequence means the restriction enzyme recognition sequence, and the underline is the complementary sequence of the CCTF produced thereby. In the examples of the present invention, the part represented by * is a tag that modified dCTP was inserted into C in the recognition sequence to block the site cleaved by the PspGI restriction enzyme.

The sequence and mass of the CCTF produced in the amplified product are as follows.

(SEQ ID. NO: 7) CCTF 1: 5′-CCAGGATAGTTCA-3′/4038.6 Da (SEQ ID NO: 8) CCTF 2: 5′-CCAGGTATCATAGAT-3′/4351.8 Da

2. PCR Amplification

Primer 1 and Primer 3 as forward primers, and Primer 2 and Primer 4 as reverse primers were subjected and PCR reaction was performed simultaneously, and then, the formation of CCTF was determined.

20

Of the total reaction solution comprising each Primer 3 μM, PspGI (NEB, USA) 2U, PCR buffer (1×), MgSO₄ 3 mM, dNTP 400 μM, Vent Polymerase (NEB, USA) 1 U and NG, MH template DNA 100 pg/ul was subjected to PCR reaction using C1000 PCR (Bio-Rad, USA) under the following conditions:

94° C. 10 mins,

94° C. 30 secs, 62° C. 30 secs 72° C. 30 secs (35 cycles),

85° C. 2.5 hours

3. Purification and Desalting of the Cleaved Fragments During the PCR Reaction

Oasis (Waters) C18 reverse phase column chromatography was used to isolate the DNA fragments cleaved by treatment with a restriction enzyme during the PCR reaction from the above solution. To the solution treated with the restriction enzyme, 70 μl of 0.15 M triethylammonium acetate (TEAA, pH 7.6) was added and allowed to stand for 1 minute. Resin was activated by passing 1 ml of 100% acetonitrile (ACN; Sigma, USA) and 0.1 M TEAA to the column, and then, 100 s of a mixed solution of the solution treated with the restriction enzyme and 0.15M TEAA, 2 ml of 0.1M TEAA and 1 ml of the third distilled water were passed through in this order. The column was placed on a Collection Plate and 100 μl of 70% ACN was passed. When the eluate was collected on the collection plate, the collection plate was dried at 120° C. for 60 minutes.

4. MALDI-TOF MS Analysis

4 μ

of MALDI matrix [22.8 mg ammonium citrate, 148.5 mg hydroxypicolinic acid, 1.12 m

acetonitrile, 7.8 m

H₂O] was previously dotting on Anchor chip plate of MALDI-TOF mass spectrometry (Biflex IV, Bruker), and then, was dried at 37° C. for 30 minutes. 10 μl of the third distilled water was dissolved in a sample of the collection plate after the purification and desalting procedure, and 2 μl of the solution was dropped onto the dried MALDI Matrix, the Maldin Matrix was dried again at 37° C. for 30 minutes, and then was analyzed by MALDI-TOF mass spectrometry. The analysis method follows the manual of the MALDI-TOF mass spectrometry.

The result of analyzing the CCTF produced by the above reaction using a mass spectrometer is as shown in FIG. 2. From the result of FIG. 2, it can be confirmed the peaks of 4083 Da, the mass of CCTF 1 which can be formed when performing PCT with the combination of Primer 1 and Primer 2, and 4351 Da, the mass of CCTF 2 which can be formed when performing PCT with the combination of Primer 3 and Primer 4 (a). These results demonstrated that the PCR amplification product can be analyzed using CCTF formed by CTPO, and that CCTF can be used to accurately amplify and differentiate the target sequence in the reaction product comprising various primers.

Therefore, it was demonstrated that the target nucleic acid sequence can be detected more precisely than the conventional PCR method by performing the PCR using the CCTF marking technique and distinguishing the tag fragments of various lengths through mass analysis using MALDI-TOF MS after performing PCR.

Example 2. Formation of CCTF and Analysis of Inherent Dissociation Temperature Peak of CCTF in Multiple Target PCR

The CCTF generated during the PCR reaction is combined with the SCO capable of generating a fluorescence signal at the inherent dissociation temperature to form an intrinsic dissociation temperature peak, which can be observed directly after the PCR process using a real-time PCR instrument. During the PCR reaction, CCTF is formed, and at the same time it is hybridized with the CCTF complementary sequence region of SCO to form a double strand. By measuring the inherent dissociation temperature of SCO seen when the double strand is dissociated into a single strand, the kinds of CCTF can be discriminated and analyzed simultaneously with PCR through a real-time PCR instrument. The SCO used in this example used different fluorescent reporters, respectively, and the inherent dissociation temperature was adjusted to enable discrimination of CCTF.

In this example, CCTF analysis was performed using a real-time PCR instrument using 12 kinds of the causative organisms of the sexually transmitted diseases, 5 types of the causative organisms of gastrointestinal diseases, 9 types of HPV subtypes, 10 types of the causative organisms of the respiratory disease and single base mutation rs6265 nucleic acid of BDNF gene, respectively.

1. Formation of CCTF in multi-target PCR of the causative organisms of the sexually transmitted diseases and analysis of the inherent dissociation temperature peak of CCTF

CCTF analysis for Chlamydia trachomatis(CT), Neisseria. gonorrhea (NG) Mycoplasma hominis(MH), Mycoplasma genitalium(MG), Trichomonas vaginalis(TV), Ureaplasma urealyticum(UU), Ureaplasma parvum(UP), Candida albicans(CA), Gardnerella vaginalis(GV), Herpes simplex virus 1 (HSV 1), Herpes simplex virus 2 (HSV 2), Treponema pallidum (TP), the causatives agents of sexually transmitted diseases and Internal control (IC) DNA was performed using Real-time PCR instrumentation.

1) Primer for Target Sequence Template DNA Constructed by the Sequence-Specific Manner

The forward primer used in this example was CTPO and was constructed on the same principle as in Example 1 above. The 5′end of CTPO was composed of 19-20 mers of nucleotide sequences, and was composed of a sequence non-complementary to DNA of the target sequence to form CCTF. The restriction enzyme recognition sequence was then located, and from this up to the 3′ end, it was composed of the sequence complementary to each target site was composed to play a role as a primer. The reverse primer was composed of sequence complementary to the target site to be amplified.

In addition, SCO, which forms a complementary bond with CCTF to be a double-stranded template, was positioned by positioning fluorescent offsetting molecules (BHQ-1 or BHQ-2), and the fluorescent reporter molecular was positioned so as to have a certain distance.

Primer information and target sequence information which is amplified and generated are as follows

Primer 5: (SEQ ID. NO: 9) 5′- CCACTCCAGCCGGCTGACA*CCAGGACTTGGTGTGACGCTATCAGCAT- 3′ Primer 6: (SEQ ID. NO: 10) 5′-GTTTTCAAAACACGGTCGAAAACAAAGTC-3′ Primer 7: (SEQ ID. NO: 11) 5′- CATCGCCACGAGCCGGTTAA*CCAGGTTGAAACACCGCCCGGAACCC-3′ Primer 8: (SEQ ID. NO: 12) 5′-GCTCCTTATTCGGTTTGACCGGT-3′ Primer 9: (SEQ ID. NO: 13) 5′- ACTCACGCTAATGGAGCGCA*CCAGGTTTAGCTCCTATTGCCAACGTATT GG-3′ Primer 10: (SEQ ID. NO: 14) 5′-TGTGTGGAGCATCTTGTAATCTTTGGTC-3′ Primer 11: (SEQ ID. NO: 15) 5′- GCTACCCAGCCGGCTACAAG*CCAGGCTTTATGGTGCTTATATTGGTGGC ATG-3′ Primer 12: (SEQ ID. NO: 16) 5′-CTGTATAACGTTGTGCAGCAGGTC-3′ Primer 13: (SEQ ID. NO: 17) 5′- TGCCGCGTGATTCGATCCCA*CCAGGTATGTCCGGCACAACATGCGCT- 3′ Primer 14: (SEQ ID. NO: 18) 5′-GAGGCTTACGAAGGTCGGAGTTGA-3′ Primer 15: (SEQ ID. NO: 19) 5′- TCTCATAGCTGGGCCGCTG*CCAGGAAGTAGCATATGATGAAGCACACAA CA-3′ Primer 16: (SEQ ID. NO: 20) 5′-TAATGCAACGTGCATTTGCTTCAAC-3′ Primer 17: (SEQ ID. NO: 21) 5′- CAGATCGTTGGCACTCTGCGA*CCAGGTTAAAGTAGCATATGATCAAGCT CATTCA-3′ Primer 18: (SEQ ID. NO: 22) 5′-TTGTAATGATACAACGAGCATCATCATTAAT-3′ Primer 19: (SEQ ID. NO: 23) 5′- GCTCGTATGCCGCTCCATATA*CCAGGCCAAATCTGGATCTTCCTCTGCA TC-3′ Primer 20: (SEQ ID. NO: 24) 5′-GAGCTTGAGCTGGACCCAGAG-3′ Primer 21: (SEQ ID. NO: 25) 5′- ACGTGCCGTGCATCGTTGCA*CCAGGCAACCGGCTCCATTTTGGTGGAG- 3′ Primer 22: (SEQ ID. NO: 26) 5′-CGTCACGTCCTTCATCGGTCC-3′ Primer 23: (SEQ ID. NO: 27) 5′- TCGCAGTCCCGTCGAGGAA*CCAGGAGGCCTGGCTATCCGGAGAAAC-3′ Primer 24: (SEQ ID. NO: 28) 5′-CGTTGTGTTGGCCGCAGGTC-3′ Primer 25: (SEQ ID. NO: 29) 5′- CTCATAGCTAGGCGCCTG*CCAGGGCTGCACGTGGGTCTGTTGTG-3′ Primer 26: (SEQ ID. NO: 30) 5′-GGAAACGCAGGCCACGAAACC-3′ Primer 27: (SEQ ID. NO: 31) 5′-GCTTCGCGTCTCAGGCCTGT*CCAGGGGGCATTACAGTTTTGCGTCA TGAC-3′ Primer 28: (SEQ ID. NO: 32) 5′-CAAGTCTGAGCACTTGCACCG-3′ Primer 29: (SEQ ID. NO: 33) 5′- CTGTTAGCTCTGCGAGCT*CCAGGGGAGCGACACTTGTTGGTGTTGAC- 3′ Primer 30: (SEQ ID. NO: 34) 5′-TGATGAAATGAAGCCACCCGTGC-3′ SCO 1: (SEQ ID. NO: 35) TCGGAGCCAGCGCGGCGTAAAC[T(FAM)]CCACTCCAGCCGGCTGACA [BHQ1] SCO 2: (SEQ ID. NO: 36) TACAACAGCAGTACGGAGACGAC[T(HEX)]CATCGCCACGAGCCGGTTA A[BHQ1] SCO 3: (SEQ ID. NO: 37) ATTTATTCTTACTCGATGTTAAA[T(HEX)]ACTCACGCTAATGGAGCGC A[BHQ1] SCO 4: (SEQ ID. NO: 38) TATATATATATATTATTATAAA[T(CalRed610)]GCTACCCAGCCGGC TACAAG[BHQ2] SCO 5: (SEQ ID. NO: 39) AAGAATAACTACTACAATCTACT[T(Quasar670)]TGCCGCGTGATTC GATCCCA[BHQ2] SCO 6: (SEQ ID. NO: 40) TTATTATTATTATTATTATATA[T(CalRed610)]TCTCATAGCTGGGC CGCTG[BHQ2] SCO 7: (SEQ ID. NO: 41) AATCTTCAATGCTTACCGTA[T(FAM)]CAGATCGTTGGCACTCTGCGA [BHQ1] SCO 8: (SEQ ID. NO: 42) AAAATAAATAATATAATATA[T(FAM)]GCTCGTATGCCGCTCCATATA [BHQ1] SCO 9: (SEQ ID. NO: 43) TCGGAGCCAGCGCGGCGTAACG[T(Quasar670)]ACGTGCCGTGCATC GTTGCA[BHQ2] SCO 10: (SEQ ID. NO: 44) AAGAATAACTACTACAATCTAC[T(Quasar705)]TTCGCAGTCCCGTC GAGGAA[BHQ2] SCO 11: (SEQ ID. NO: 45) TCGGAGCCAGCGCGGCGTAA[T(Quasar705)]CTCTCATAGCTAGGCG CCTG[BHQ2] SCO 12: (SEQ ID. NO: 46) AAAATAAATAATATAATATAG[T(Quasar705)]CTTCGCGTCTCAGGC CTGT[BHQ2] SCO 13: (SEQ ID. NO: 47) AAAATAAATAATATAATATA[T(Quasar670)]TCTGTTAGCTCTGCGA GCT[BHQ2] Amplified product 3: GenBank: X52557.1/Position (start-end): 157-227

(SEQ ID. NO: 48) CCACTCCAGCCGGCTGACA* 

 ACTTGGTGTGACGCTATCAGCAT GCGTATGGGTTACTATGGTGACTTTGTTTTCGACCGTGTTTTGAAAAC Amplified product 4: GenBank: X52364.1/Position (start-end): 375-459

(SEQ ID. NO: 49) CGCCCACCGCATCCCGCGCCCCTCCCTCAGCA* 

 TTGAAACACC GCCCGGAACCCGATATAATCCGCCCTTCAACATCAGTGAAAATCTTTTTT TAACCGGTCAAACCGAATAAGGAGC Amplified product 5: GenBank: AJ243692.1/Position (start-end): 835-944

(SEQ ID. NO: 50) ACTCACGCTAATGGAGCGCA* 

 TTTAGCTCCTATTGCCAACGTA TTGGAAAAAAACTTTGGTATTGAAAAAGGATTTATGACAACAGTCCACTC ATATACAGCAGACCAAAGATTACAAGATGCTCCACACA Amplified product 6: GenBank: U09251.1/Position (start-end): 3462-3687

(SEQ ID. NO: 51) GCTACCCAGCCGGCTACAAG* 

 CTTTATGGTGCTTATATTGGTG GCATGCACCATGATCGTCCTTTTAAAAAGTCTGCGAGGATTGTTGGTGAT GTAATGAGTAAATTCCACCCTCATGGTGATATGGCAATATATGACACCAT GTCAAGAATGGCTCAAGACTTTTCATTAAGATACCTTTTAATTGATGGTC ATGGTAATTTTGGTTCTATAGATGGTGATAGACCTGCTGCACAACGTTAT ACAG Amplified product 7: GenBank: XM_001582993.1/Position (start-end): 705-768

(SEQ ID. NO: 52) TGCCGCGTGATTCGATCCCA* 

 TATGTCCGGCACAACATGCGCT TATGTCCGGCACAACATGCGCTCTCCGCTTCCCAGGTCAGCTCAACTCCG ACCTTCGTAAGCTC Amplified product 8: GenBank: AF085700.2/Position (start-end): 4673-4873

(SEQ ID. NO: 53) TCTCATAGCTGGGCCGCTG* 

 AAGTAGCATATGATGAAGCACAC AACAAAATGGCGCATACTGTGTATTTCACTAATTTCTATCGTTCATCAAA ACCACTATTTTTAGATGAAGAAGACCCAATTAATCCCTGTTTTCAAACTA TTAGTATGGGTGGGGGTTATGTATCTGGTGAAGTGTATCGTTCTGATTTT GAAGTTGAAGCAAATGCACGTTGCATTA Amplified product 9: GenBank: AF085733.2/Position (start-end): 4677-4886

(SEQ ID. NO: 54) CAGATCGTTGGCACTCTGCGA* 

 TTAAAGTAGCATATGATCAAG CTCATTCAAAAATGGCACATACTGTCTATTTTACGAATTTTTATCGTTCA TCTAAACCTTTATTTTTAGATGAAGAAGATCCAATCAACCCCTGTTTTCA AACAATTAGTATGGGTGGTGGATATGTTTCAGGTGAAATTTATCGTTCTG ATTTTGAAATTAATGATGATGCTCGTTGTATCATTACAA Amplified product 10: GenBank: M90812.1/Position start-end): 1736-1811

(SEQ ID. NO: 55) GCTCGTATGCCGCTCCATATA* 

 CCAAATCTGGATCTTCCTCTG CATCTGCTTCTGGATCATCAAGCAGCAGCACCAGCTCTGGGTCCAGCTCA AGCTC Amplified product 11: GenBank: L08167.1/Position (start-end): 273-434

(SEQ ID. NO: 56) ACGTGCCGTGCATCGTTGCA* 

 CAACCGGCTCCATTTTGGTGGA GTCGCTTGATCGTTTTGTGATCGTTTAGTGTGATGATTTATTATGTCTAG AGAGTTAAGCGATAGGCTTTTACTGGTGTATCACTGTAAGGGCGTATTGG TTGGATGCCTTGGTAGACAGGACCGATGAAGGACGTGACG Amplified product 12:DQ889502.1/Position (start-end): 123860-124007

(SEQ ID. NO: 57) TCGCAGTCCCGTCGAGGAA* 

 AGGCCTGGCTATCCGGAGAAACA GCACACGACTTGGCGTTCTGTGTGTCGCGATGTCTCTGCGCGCAGTCTGG CATCTGGGGCTTTTGGGAAGCCTCGTGGGGGCTGTTCTTGCCGCCACCCA TCGGGGACCTGCGGCCAACACAACG Amplified product 13: GenBank: EU018100.1/Position (start-end): 561-746

(SEQ ID. NO: 58) CTCATAGCTAGGCGCCTG* 

 GCTGCACGTGGGTCTGTTGTGGGT AGAGGTGGGCGGGGAGGGCCCCGGCCCCACCGCCCCCCCCACAGGCGGCG CGTGCGGAGGGCGGCCCGTGCGTCCCCCCGGTCCCCGCGGGCCGCCCGTG GCGCTCGGTGCCCCCGGTATGGTATTCCGCCCCCAACCCCGGGTTTCGTG GCCTGCGTTTCC Amplified product 14: GenBank: U57757.1/Position (start-end): 910-1067

(SEQ ID. NO: 59) GCTTCGCGTCTCAGGCCTGT* 

 GGGCATTACAGTTTTGCGTCAT GACGGCTTTGAAGCTGACGACCTCATTGCAACCCTAGCAAAACGAGTTGC GGCTGAGCACTGTCATGTTGTGATTATCTCCTCAGATAAAGATGTACTTC AGCTTGTGTGTGATACGGTGCAAGTGCTCAGACTTG Amplified product 15: GenBank: NM 001035551.2/Position (start-end): 214-369

(SEQ ID. NO: 60) CTGTTAGCTCTGCGAGCT* 

GGAGCGACACTTGTTGGTGTTGACA AGTTCGGTAACAAATACTACCAGAAGCTAGGCGATACTCAATACGGTATG CACAGATGGGTAGAGTATGCTTCAAAGGATCGTTACAACGCATCTCAAGT ACCAGCTGAATGGCACGGGTGGCTTCATTTCATCA

The bold and slanted font of the Primer sequences means the restriction enzyme recognition sequence, and the underline is the complementary sequence of the CCTF produced thereby. the part represented by * is a tag that modified dCTP was inserted into C in the recognition sequence to block the site cleaved by the PspGI restriction enzyme. In SCO, the parentheses mean the position of the nucleotide sequence in which the fluorescent offsetting molecule and the fluorescent reporter are located. The sequence of the CCTF produced from the amplified product is as follows.

CCTF 3: (SEQ ID. NO: 61) 5′- CCTGGTGTCAGCCGGCTGGAGTGG -3′ CCTF 4: (SEQ ID. NO: 62) 5′- CCTGGTTAACCGGCTCGTGGCGATG -3′ CCTF 5: (SEQ ID. NO: 63) 5′- CCTGGTGCGCTCCATTAGCGTGAGT -3′ CCTF 6: (SEQ ID. NO: 64) 5′- CCTGGCTTGTAGCCGGCTGGGTAGC -3′ CCTF 7: (SEQ ID. NO: 65) 5′- CCTGGTGGGATCGAATCACGCGGCA -3′ CCTF 8: (SEQ ID. NO: 66) 5′- CCTGGCAGCGGCCCAGCTATGAGA -3′ CCTF 9: (SEQ ID. NO: 67) 5′- CCTGGTCGCAGAGTGCCAACGATCTG -3′ CCTF 10: (SEQ ID. NO: 68) 5′- CCTGGTATATGGAGCGGCATACGAGC -3′ CCTF 11: (SEQ ID. NO: 69) 5′- CCTGGTGCAACGATGCACGGCACGT -3′ CCTF 12: (SEQ ID. NO: 70) 5′- CCTGGTTCCTCGACGGGACTGCGA -3′ CCTF 13: (SEQ ID. NO: 71) 5′- CCTGGCAGGCGCCTAGCTATGAG -3′ CCTF 14: (SEQ ID. NO: 72) 5′- CCTGGACAGGCCTGAGACGCGAAGC -3′ CCTF 15: (SEQ ID. NO: 73) 5′- CCTGGAGCTCGCAGAGCTAACAG -3′

2) PCR Amplification and Determination of SCO Inherent Dissociation Temperature

-   -   PCR reaction was performed using the following CFX96 Real-time         PCR (Bio-Rad, USA) with 20 μ         of total reaction solution of each of Primer 5, 6, 7, 8, 9, 10,         11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,         27, 28, 29, 30 and SCO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13         prepared by adding 0.15 μM, PspGI (NEB, USA) 51, PCR buffer         (1×), MgCl₂ 2.5 mM, dNTP 200 μM, h-Taq DNA polymerase (Solgent,         Korea) 1.6 U, template DNA of genomic DNA of CT, NG, MH, MG, TV,         UU, UP, CA, GV, HSV1, HSV2, TP and IC 100 pg/rxn, respectively.

95° C. 15 mins,

95° C. 30 secs, 63° C. 1 min (50 cycles).

A reaction was performed using a cycle at the denaturation temperature of 95° C. for 15 minutes once, and with a cycle at the denaturation temperature of 95° C. for 30 seconds, and an annealing temperature of 63° C. for 1 minute 50 times. After the reaction, the reaction mixture was cooled to 50° C. in the same apparatus, held at 50° C. for 30 seconds, and then slowly heated from 50° C. to 95° C. to obtain an inherent dissociation temperature analysis curve. Data analysis was performed with Bio-Rad CFX Manager 1.6.

FIG. 3, (a) shows the results of multiple inherent dissociation temperature measurements for causative organisms of CT, NG, MH, MG, TV, UU, UP, CA, GV, HSV1, HSV2, TP, IC. The peak was observed at the inherent dissociation temperature that each SCO has (CT: FAM 80° C., NG: HEX 76.5° C., MH: HEX 68° C., MG: CaRed610 67.5° C., TV: Quasar670 71.5° C., UU: CaRed610 77° C., UP: FAM 77° C., CA: FAM 65° C., GV: Quasar670 78.5° C., HSV 1: Quasar705 73.5° C., HSV 2: Quasar705 79° C., TP: Quasar705 66° C., IC: Quasar670 63.5° C.) (a)(b)(c)(d)(e)(f), and no peak of SCO visualizing CCTF was observed when the target sequence was not added in the same composition (g).

Therefore, it was proved that the target nucleic acid sequence can be detected more quickly and simply than the conventional PCR method by analyzing the fluorescence of the SCO using the real-time PCR instrument, simultaneously with the PCR using the marking technique of CCTF.

2. Formation of CCTF in Multi-Target PCR of the Causative Organism for the Gastrointestinal Diseases and Analysis for the Inherent Dissociation Temperature Peak of CCTF

CCTF analysis was performed with Real-time PCR instrument for DNA of the causative organisms of the gastrointestinal diseases, Rotavirus A(RVA), Astrovirus(AstV), Adenovirus F40(AdV 40), Adenovirus F41(AdV 41), Norovirus GI(NoV GI), Norovirus GII(NoV GII), and External control (EC).

1) Primer for the Target Sequence of Template DNA Constructed in the Sequence-Specific Manner

The forward primer used in this example was CTPO and was constructed on the same principle as in Example 1 above. The 5′end of CTP was composed of 19-20 mers of nucleotide sequences, and was composed of a sequence non-complementary to DNA of the target sequence so as to form CCTF. The restriction enzyme recognition sequence was consecutively located, and after this up to the 3′ end, it was composed of the sequence complementary to each target site to play a role as a primer. The reverse primer was composed of sequence complementary to the target site to be amplified.

In addition, SCO, which forms a complementary bond with CCTF to be a double-stranded template, was positioned by positioning fluorescent offsetting molecules (BHQ-1 or BHQ-2), and the fluorescent reporter molecular was positioned so as to have a certain distance.

Primer information and target sequence information which is amplified and generated are as follows.

Primer 31: (SEQ ID. NO: 74) 5′-GCAGGAGCCTCTCATCTCG*CCAGGCTCATTTATAGACARCTTCTCA CTAATTC-3′ Primer 32: (SEQ ID. NO: 75) 5′-AGTTTTTTCTGATCCAATYTGYTCTATTTC-3 Primer 33: (SEQ ID. NO: 76) 5′-TCAGACGGTTCGAGGCTCC*CCAGGARGATYAAGCGTGGAGTATAYA TGG-3′ Primer 34: (SEQ ID. NO: 77) 5′-TTTGCGTGCYTCTTCACACGC-3′ Primer 35: (SEQ ID. NO: 78) 5′-AACGCGAATCGACCGGAT*CCAGGCGCGATGTGTTTGCCGATAAAA C-3′ Primer 37: (SEQ ID. NO: 79) 5′-CATTGCGTCTGCCECACTTG-3′ Primer 38: (SEQ ID. NO: 80) 5′-AACGCGAATCGACCGGAT*CCAGGAAACAAGAACACCTATGCCTACA TGAAC-3′ Primer 39: (SEQ. ID. NO: 81) 5′-ATGTTAACGTCCTTCCTGAAGTTCCAC-3 Primer 40: (SEQ ID. NO: 82) 5′-TAGATCGGACTGCGAATCG*CCAGGGAGATCGCRATCTYCTGCCCGA -3 Primer 41: (SEQ ID. NO: 83) 5′-RGCGTCCTTAGACGCCATCATC-3 Primer 42: (SEQ ID. NO: 84) 5′-ATCTACAGCGTCGCATCACG*CCAGGCGCAATCTGGCTCCCARTTTT GTG-3 Primer 43: (SEQ ID. NO: 85) 5′-GCGTCAYTCGACGCCATCYTCA-3 Primer 44: (SEQ ID. NO: 86) 5′-CATAGGTCGAGGTCCTCAC*CCAGGGCAAACTCCGGCATCTACTAAT AGACG-3 Primer 45: (SEQ ID. NO: 87) 5′-AAGCGGTGATCCGCACAGTG-3 SCO 14: (SEQ ID. NO: 88) TCGGCCGATCGTCCATAGAGTCAAGC[T(HEX)]CGCAGGAGCCTCTCAT CTCG[BHQ1]  SCO 15: (SEQ ID. NO: 89) TCACGATGAGCGAGTTGAGCTACG[T(Calred610]ATCAGACGGTTCG AGGCTCC[BHQ2] SCO 16: (SEQ ID. NO: 90) TGTTCAATATATAATGATAATATG[T(Calred610)]AACGCGAATCGA CCGGAT[BHQ2] SCO 17: (SEQ ID. NO: 91) TGTTCAATATATAATGATAATATG[T(Calred610)]AACGCGAATCGA CCGGAT[BHQ2] SCO 18: (SEQ ID. NO: 92) ACATTTATAATACAGTATTTTA[T(FAM)]TAGATCGGACTGCGAATCG [BHQ1] SCO 19: (SEQ ID. NO: 93) AGCTCCTGCCAGTACTGCCATCCA[T(FAM)]ATCTACAGCGTCGCATCA CG[BHQ1] SCO 20: (SEQ ID. NO: 94) TAGTTATAATGAATAACTATTAT[T(HEX)]CATAGGTCGAGGTCCTCA C[BHQ1] Amplified product 16: GenBank: KT694942.1/Position (start-end): 19-99

(SEQ ID NO: 95) GCAGGAGCCTCTCATCTCG *

CTCATTTATAGACARCTTCTCACT AATTCATATTCAGTAGATTTACATGATGAAATAGARCARATTGGATCAGA AAAAACT Amplified product 17: GenBank: AB000287.1/Position (start-end): 2232-2321

(SEQ ID NO: 96) TCAGACGGTTCGAGGCTCC *

ARGATYAAGCGTGGAGTATAYATG GACCTGCTTGTCTCGGGGGCAAGCCCAGGCAATGCATGGTCCCATGCGTG TGAAGARGCACGCAAA Amplified product 18: GenBank: KM274923.1/Position (start-end): 121-179

(SEQ. ID NO: 97) AACGCGAATCGACCGGAT* 

CGCGATGTGTTTGCCGATAAAACGT CACAACCGGAGCCCCAAGTGGGGCAGACGCAATG Amplified product 19: GenBank: AB330122.1/Position (start-end): 1407-1691

(SEQ ID. NO: 98) AACGCGAATCGACCGGAT *

AAACAAGAACACCTATGCCTACATG AACGGTCGGGTGGCGGTTCCTAGCGCCCTCGATACCTACGTAAACATCGG GGCACGGTGGTCTCCAGATCCCATGGACAATGTTAACCCCTTCAATCACC ACCGTAACGCCGGTCTGCGCTATCGATCCATGCTCTTUGGCAACGGGCGT TACGTACCCTTCCACATTCAAGTCCCCCAGAAGTTTTTTGCCATTAAAAA TCTCCTCCTCTTACCGGGTTCCTACACCTACGAGTGGAACTTCAGGAAGG ACGTTAACAT Amplified product 20: GenBank: LN854564.1/Position (start-end): 5325-5378

(SEQ ID NO: 99) TAGATCGGACTGCGAATCG *

GAGATCGCRATCTYCTGCCCGAAT TCGTAAATGATGATGGCGTCTAAGGACGCY Amplified product 21: GenBank: KT202798.1/Position (start-end): 5060-5107

(SEQ ID. NO: 100) ATCTACAGCGTCGCATCACG *

CGCAATCTGGCTCCCARTTTTGT GAATGARGATGGCGTCGARTGACGC Amplified product 22: GenBank: EF204940.1/Position (start-end): 1707-1878

(SEQ ID. NO: 101) CATAGGTCGAGGTCCTCAC* 

GCAAACTCCGGCATCTACTAATAG ACGCCGGCCATTCAAACATGAGGATTACCCATGTCGAAGACAACAAAGAA GTTCAACTCTTTATGTATTGATCTTCCTCGCGATCTTTCTCTCGAAATTT ACCAATCAATTGCTTCTGTCGCTACTGGAAGCGGTGATCCGCACAGTG

The bold and slanted font of the Primer sequence means the restriction enzyme recognition sequence, and the underline is the complementary sequence of the CCTF produced thereby. the part represented by * is a tag that modified dCTP was inserted into C in the recognition sequence to block the site cleaved by the PspGI restriction enzyme. In SCO, the parentheses mean the position of the nucleotide sequence in which the fluorescent offsetting molecule and the fluorescent reporter are located. The sequence of the CCTF produced from the amplified product is as follows.

CCTF 16: (SEQ ID. NO: 102) 5′- CCTGGTGTCAGCCGGCTGGAGTGG3′ CCTF 17: (SEQ ID. NO: 103) 5′- CCTGGTTAACCGGCTCGTGGCGATG3′ CCTF 18: (SEQ ID. NO: 104) 5′- CCTGGTGCGCTCCATTAGCGTGAGT3′ CCTF 19: (SEQ ID. NO: 105) 5′- CCTGGCTTGTAGCCGGCTGGGTAGC -3′ CCTF 20: (SEQ ID. NO: 106) 5′- CCTGGTGGGATCGAATCACGCGGCA -3′ CCTF 21: (SEQ ID. NO: 107) 5′- CCTGGCAGCGGCCCAGCTATGAGA -3′ CCTF 22: (SEQ ID. NO: 108) 5′- CCTGGTCGCAGAGTGCCAACGATCTG -3′ CCTF 23: (SEQ ID. NO: 109) 5′- CCTGGTATATGGAGCGGCATACGAGC -3′ CCTF 24: (SEQ ID. NO: 110) 5′- CCTGGTGCAACGATGCACGGCACGT -3′ CCTF 25: (SEQ ID. NO: 111) 5′- CCTGGTTCCTCGACGGGACTGCGA -3′ CCTF 26: (SEQ ID. NO: 112) 5′- CCTGGCAGGCGCCTAGCTATGAG -3′ CCTF 27: (SEQ ID. NO: 113) 5′- CCTGGACAGGCCTGAGACGCGAAGC -3′ CCTF 28: (SEQ ID. NO: 114) 5′- CCTGGAGCTCGCAGAGCTAACAG -3′

2) PCR Amplification and Determination of the Inherent Dissociation Temperature of SCO

The following PCR reaction was performed using CFX96 Real-time PCR (Bio-Rad, USA) with 20 μ

of total reaction solution of each of Primer 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and SCO 14, 15, 16, 17, 18, 19, 20 prepared by adding 0.15 μM, PspG (NEB, USA) 1U, PCR buffer (1×), MgCl₂ 2.5 mM, dNTP 200 μM, DTT 0.1 mM, RNase Inhibitor IU, SuperiorScript II (Enzynomics, Korea) 1U and the nucleic acid of the genomic RNA of RVA, AstV, AdV 40, AdV41, NoV GI, NoV GII and EC(MS2 phage) 1×10{circumflex over ( )}⁴ pg/rxn, respectively.

55° C. 20 mins, 95° C. 10 mins

95° C. 30 secs, 63° C. 1 mins (50 cycles).

A reverse transcription reaction was performed using a cycle at the denaturation temperature of 55° C. for 20 minutes once, and with a cycle at the denaturation temperature of 95° C. for 10 minutes 1 time, and with a cycle at an annealing temperature of 63° C. for 1 minute 50 times repeatedly. After the reaction, the reaction mixture was cooled to 50° C. in the same apparatus, held at 50° C. for 30 seconds, and then slowly heated from 50° C. to 95° C. to obtain an inherent dissociation temperature analysis curve. Data analysis was performed with Bio-Rad CFX Manager 1.6.

FIG. 4 shows the results of multiple inherent dissociation temperature measurements for causative organisms of RVA, AstV, AdV 40, AdV 41, NoV GI, NoV GII. It was identified that the peak was observed at the inherent dissociation temperature that each SCO has: RVA: HEX 78° C., AstV: CalRed60 78° C., AdV 40: CalRed610 67° C., AdV 41: CalRed610 67° C., NoV GI: FAM 68° C., NoV GII: FAM 84° C., EC: HEX 69° C. (a)(b)(c)(d), and no peak of SCO visualizing CCTF was observed when the target sequence was not added in the same composition (e).

Therefore, it was proved that the target nucleic acid sequence can be detected more quickly and simply than the conventional PCR method by analyzing the fluorescence of the SCO using the real-time PCR instrument, simultaneously with the PCR using the marking technique of CCTF.

3. Formation of CCTF and Analysis for the Inherent Dissociation Temperature Peak of CCTF in Multi-Target PCR for Detecting the Human Papillomavirus

CCTF analysis was performed with Real-time PCR instrument for DNA of subtypes of Human Papillomavirus (HPV), 16 type, 18 type, 33 type, 35 type, 51 type, 53 type, 59 type, 68a type, 82 type and Internal control (IC).

1) Primer of the Target Sequence Template DNA, Constructed in the Sequence-Specific Manner

The forward primer used in this example was CTPO and was constructed on the same principle as in Example 1 above. The 5′end of CTPO was composed of 19-20 mers of nucleotide sequences, and was composed of a sequence non-complementary to DNA of the target sequence to form CCTF. The restriction enzyme recognition sequence was consecutively located, and after this up to the 3′ end, a sequence complementary to each target site was composed to play a role as a primer. The reverse primer was composed of sequence complementary to the target site to be amplified.

In addition, SCO, which forms a complementary bond with CCTF to be a double-stranded template, was positioned by positioning fluorescent offsetting molecules (BHQ-1 or BHQ-2), and the fluorescent reporter molecular was positioned so as to have a certain distance.

Primer information and target sequence information which is amplified and generated are as follows.

Primer 46: (SEG ID. NO: 115) 5′-CTCTGATAGCGACTGCTCGCA*CCAGGATAATATAAGGGGTCGGTGG ACCGG-3′ Primer 47: (SEQ ID. NO: 116) 5′-CTCCATGCATGATFACAGCTGGGTT-3′ Primer 48: (SEQ ID. NO: 117) 5′-ATCGGTCTCCTGAAAGCTGCG*CCAGGCAGAAGGTACAGACGGGGAG GGC-3′ Primer 49: (SEQ ID. NO: 118) 5′-CACCTCCAGCCGCTCCCCTAAT-3′ Primer 50: (SEQ ID. NO: 119) 5′-CTGGCGTAGAGCACTTACGCT*CCAGGCAACGATAACCGACCACCAC AAGCA-3′ Primer 51: (SEQ ID. NO: 120) 5′-CGGGGTCTGCACAGAACAGCTTT-3′ Primer 52: (SEQ ID. NO: 121) 5′-CTGGCGTAGAGCACTTACGCT*CCAGGAGGACCCAGCTGAACGACCT TACAA-3′ Primer 53: (SEQ ID. NO: 122) 5′-CTGTCCACCGTCCACCGATGTTATG-3′ Primer 54: (SEQ ID. NO: 123) 5′-CTGGCGTAGAGCACTTACGCT*CCAGGGCTGGCAACGTACACGACAA CG-3′ Primer 55: (SEQ ID. NO: 124) 5′-GCTGTACAACGCGAAGGGTGTC-3′ Primer 56: (SEQ ID. NO: 125) 5′-CTGGCGTAGAGCACTTACGCT*CCAGGTCCACCTATGCACCGAAACC TCCAA-3′ Primer 57: (SEQ ID. NO: 126) 5′-TGCAGTGACGAGTCCCCGTGTAGTA-3′ Primer 58: (SEQ ID. NO: 127) 5′-CTGGCGTAGAGCACTTACGCT*CCAGGGACTGTACACCGTATGCAGC GTG-3′ Primer 59: (SEQ ID. NO: 128) 5′-GCGTATCAGCAGCTCATGTAA-3′ Primer 60: (SEQ ID. NO: 129) 5′-CTGGCGTAGAGCACTTACGCT*CCAGGACAAACTCGACGTCGTCTCG GAA-3′ Primer 61: (SEQ ID. NO: 130) 5′-CAGGTCACCACAACAAAGGCTCCGT-3′ Primer 62: (SEQ ID. NO: 131) 5′-ATCAGGACGCAGCCGGTTCT*CCAGGCCAAGGACAGGTACGGCTGTC ATC-3′ Primer 63: (SEQ ID. NO: 132) 5′-GGTGCCCTTGAGGTTGTCCAGGTG-3′ SCO 21: (SEQ ID. NO: 133) GAGACGTTTAAGTCCGCGACCGCTC[T(HEX)]CTGATAGCGACTGCTCG CA[BHQ 1] SCO 22: (SEQ ID. NO: 134) CAGGCGACGTCCATATGGTGCGCTA[T(FAM)]CGGTCTCCTGAAAGCTG CG[BHQ 2] SCO 23: (SEQ ID. NO: 135) CCCTTAGGTAACGTCTGGC[T(Qusar 670)]GGCGTAGAGCACTTACG CT[BHQ 2] SCO 24: (SEQ ID. NO: 136) AAACTTTAATTATTGTATA[T(FAM)]CAGGACGCAGCCGGTTCT [BHQ 1] Amplified product 23: GenBank: LC193821.1/Position (start-end): 480-571

(SEQ ID. NO: 137) CTCTGATAGCGACTGCTCGCA *

ATAATATAAGGGGTCGGTGGAC CGGTCGATGTATGTCTTGTTGCAGATCATCAAGAACACGTAGAGAAACCC AGCTGTAATCATGCATGGAG Amplified product 24: GenBank: KC470209.1/Position (start-end): 538-747

(SEQ ID NO: 138) ATCGGTCTCCTGAAAGCTGCG *

CACGACAGGAACGACTCCAACG ACGCAGAGAAACACAAGTATAATATTAAGTATGCATGGACCTAAGGCAAC ATTGCAAGACATTGTATTGCATTTAGAGCCCCAAAATGAAATTCCGGTTG ACCTTCTATGTCACGAGCAATTAAGCGACTCAGAGGAAGAAAACGATGAA ATAGATGGAGTTAATCATCAACATTTACCAGCCCGACG Amplified product 25: GenBank: KU298894.1/Position (start-end): 535-860

(SEQ ID. NO: 139) CTGGCGTAGAGCACTTACGCT *

ACGCCATGAGAGGACACAAGCC AACGTTAAAGGAATATGTTTTAGATTTATATCCTGAACCAACTGACCTAT ACTGCTATGAGCAATTAAGTGACAGCTCAGATGAGGATGAAGGCTTGGAC CGGCCAGATGGACAAGCACAACCAGCCACAGCTGATTACTACATTGTAAC CTGTTGTCACACTTGTAACACCACAGTTCGTTTATGTGTCAACAGTACAG CAAGTGACCTACGAACCATACAGCAACTACTTATGGGCACAGTGAATATT GTGTGCCCTACCTGTGCACAACAATAAACATCATCTACAATGGCCGATCC TGAA Amplified product 26: GenBank: M74117.1/Position (start-end): 117-509

(SEQ ID. NO: 140) CTGGCGTAGAGCACTTACGCT*

AGGACCCAGCTGAACG ACCTTACAAACTGCATGATTTGTGCAACGAGGTAGAAGAAAGC ATCCATGAAATTTGTTTGAATTGTGTATACTGCAAACAAGAAT TACAGCGGAGTGAGGTATATGACTTTGCATGCTATGATTTGTG TATAGTATATAGAGAAGGCCAGCCATATGGAGTATGCATGAAA TGTTTAAAATTTTATTCAAAAATAAGTGAATATAGATGGTATA GATATAGTGTGTATGGAGAAACGTTAGAAAAACAATGCAACAA ACAGTTATGTCATTTATTAATTACGTGTATTACATGTCAAAAA CCGCTGTCTCCAGTTGAAAAGCAAAGACATTTAGAAGAAAAAA AACGATTCCATAACATCGGTGGACGGTGGACAG Amplified product 27: GenBank: KU298905.1/Position (start-end): 512-812

(SEQ ID. NO: 141) CTGGCGTAGAGCACTTACGCT*

GCTGGCAACGTACAC GACAACGTAACGAAACCCAAGTGTAATAAAGCCATGCGTGGTAA TGTACCACAATTAAAAGATGTAGTATTGCATTTAACACCACAGA CTGAAATTGACTTGCAATGCTACGAGCAATTTGACAGCTCAGAG GAGGAGGATGAAGTAGATAATATGCGTGACCAGCTACCAGAAAG ACGGGCTGGACAGGCTACGTGTTACAGAATTGAAGCTCCGTGTT GCAGGTGTTCAAGTGTAGTACAACTGGCAGTGGAAAGCAGTGGA GACACCCTTCGCGTTGTACAGC Amplified product 28: GenBank: KU298906.1/Position (start-end): 3374-3558

(SEQ ID. NO: 142) CTGGCGTAGAGCACTTACGCT*

TCCACCTATGCACCGA AACCTCCAAGACCTCCGCATTGTCCGTGGGTGCCAAAGACACAC ACCTACAACCACCACAGAAACGACGACGACCAGACGTCACAGAC TCCAGAAACACCAAGTACCCCAACAACCTTTTGCGGGGACAACA ATCCGTGGACAGTACTACACGGGGACTCGTCACTGCA  Amplified product 29: GenBank: KU298922.1/Position (start-end): 226-366

(SEQ ID. NO: 143) CTGGCGTAGAGCACTTACGCT*

GTTAAGACCGAAAACG GTGCATATAAAGGTAGTTAGAAAGAAAAGGGCAACGGCATGGCA CGCTTTGAGGATCCTACACAACGACCATACAAACTGCCTGACTT GAGCACAACATTGAATATTCCTCTGCATGATATTCGC Amplified product 30: GenBank: KC470271.1/Position (start-end): 3389-3541

(SEQ ID. NO: 144) CTGGCGTAGAGCACTTACGCT*

ATGGCGCTATTTCAC AACCCTGAGGAACGGCCATACAAATTGCCAGACCTGTGCAGGA CATTGGACACTACATTGCATGACGTTACAATAGAGTGTGTCTA TTGCAGAAGGCAACTACAACGGACAGAGGTATATGAATTTGCC TTTAGTGAC  Amplified product 31: GenBank: EF450778.1/Position (start-end): 431-681

(SEQ ID. NO: 145) GCTCATATGCGGCGCCATTTA*

GCAGGTTGCTATCAAG GTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTG GAGACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCTCT CTGCCTATTGGTCTATTTTCCCACCCTTAGGCTGCTGGTGGTCT ACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCC ACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGG CAAGAAAGTGCTCGG

The bold and slanted font of the Primer sequence means the restriction enzyme recognition sequence, and the underline is the complementary sequence of the CCTF produced thereby. the part represented by * is a tag that modified dCTP was inserted into C in the recognition sequence to block the site cleaved by the PspGI restriction enzyme. In SCO, the parentheses mean the position of the nucleotide sequence in which the fluorescent offsetting molecule and the fluorescent reporter are located. The sequence of the CCTF produced from the amplified product is as follows.

CCTF 29: (SEQ ID. NO: 146) 5′-TGCGAGCAGTCGCTATCAGAG-3′ CCTF 30: (SEQ ID. NO: 147) 5′-CGCAGCTTTCAGGAGACCGAT-3′ CCTF 31: (SEQ ID. NO: 148) 5′-AGCGTAAGTGCTCTACGCCAG-3′ CCTF 32: (SEQ ID. NO: 149) 5′-AGAACCGGCTGCGTCCTGAT-3′

2) PCR Amplification and Determination of the Inherent Dissociation Temperature of SCO

The following PCR reaction was performed using CFX96 Real-time PCR (Bio-Rad, USA) with 20 μ

of total reaction solution of each of Primer 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 and SCO 21, 22, 23, 24 prepared by adding 0.15 μM, PspGI (NEB, USA) 5U, PCR buffer (1×), MgCl₂ 2.5 mM, dNTP 2 μM, h-Taq DNA polymerase (Solgent, Korea) 1.6 U and HPV type 16, type 18, type 33, type 35, type 51, type 53, type 59, type 68a, type 82 and template DNA of genomic DNA of IC 100 μg/rxn, respectively.

95° C. 15 mins,

95° C. 30 secs, 63° C. 1 mins (50 cycles).

A reaction was performed using a cycle at the denaturation temperature of 95° C. for 15 minutes once, and with a cycle at the denaturation temperature of 95° C. for 30 second and at an annealing temperature of 63° C. for 1 minute 50 times repeatedly. After the reaction, the reaction mixture was cooled to 50° C. in the same apparatus, held at 50° C. for 30 seconds, and then slowly heated from 50° C. to 95° C. to obtain an inherent dissociation temperature analysis curve. Data analysis was performed with Bio-Rad CFX Manager 1.6.

FIG. 5 shows the results of multiple inherent dissociation temperature measurements for each target of type 16, type 18, type 33, type 35, type 51, type 53, type 59, type 68a, type 82, IC. It was identified that the peak was observed at the inherent dissociation temperature that each SCO has (type 16: HEX 76.5° C., type 18: FAM 78° C., type 33: Quasar670 71° C., type 35: Quasar670 71C, type 51: Quasar670 71° C. type 53: Quasar670 71° C., type 59: Quasar670 71PC, type 68 a: Quasar670 71° C., type 82: Quasar670 71° C., IC: Quasar670 67.5° C.) (a)(b)(c)(d), and no peak of SCO visualizing CCTF was observed when the target sequence was not added in the same composition (e).

Therefore, it was proved that the target nucleic acid sequence can be detected more quickly and simply than the conventional PCR method by analyzing the fluorescence of the SCO using the real-time PCR instrument, simultaneously with the PCR using the marking technique of CCTF.

4. Formation of CCTF and Analysis for the Inherent Dissociation Temperature Peak of CCTF in Multi-Target PCR for Detecting the Causative Organism of the Respiratory Diseases

CCTF analysis was performed using Real-time PCR instrument of nucleic acids of the causative organisms of the respiratory diseases, Influenza A/H1N1(Flu A/H1N1), Influenza A/H3N2(Flu A/H3N2), Influenza A/H1N1/2009pdm(Flu A/H1N1/2009pdm), Influenza B(Flu B), Parainfluenza 1(PIV1), Parainfluenza 3(PIV3), Respiratory syncytial virus A(RSV A), Respiratory syncytial virus B(RSV B), Human metapneumovirus(MPV), Adenovirus(AdV) and External control (EC).

1) Primer for the Target Sequence of Template DNA, Constructed in the Sequence-Specific Manner

The forward primer used in this example was CTPO and was constructed on the same principle as in Example 1 above. The 5′end of CTPO was composed of 19˜20 mers of nucleotide sequences, and was composed of a sequence non-complementary to DNA of the target sequence so as to form CCTF. The restriction enzyme recognition sequence was then consecutively located, and after this up to the 3′ end, a sequence complementary to each target site was composed to play a role as a primer. The reverse primer was composed of sequence complementary to the target site to be amplified.

In addition, SCO, which forms a complementary bond with CCTF to be a double-stranded template, was positioned by positioning fluorescent offsetting molecules (BHQ-1 or BHQ-2), and the fluorescent reporter molecular was positioned so as to have a certain distance.

Primer information and target sequence information which is amplified and generated are as follows.

Primer 64:  (SEQ ID. NO: 150) 5′-TTGCTATGGCTGACGGGGAAGAATGG-3′ Primer 65: (SEQ ID. NO: 151) 5′-GCCCCGTTGAGAGCACGAAT*CCAGGG GGGTGAATCTTCTGCTTAATGTGAAGACAC-3′ Primer 66: (SEQ ID. NO: 152) 5′-GGGCACCATGCAGTACCAAACGGAAC-3′ Primer 67: (SEQ ID. NO: 153) 5′-CCGTGGCGCGAACTTATCGA*CCAGGATC ACACTGAGGGTCTCCCAATAGAGC-3′ Primer 68: (SEQ ID. NO: 154) 5′-TCAAAGACTAAGTGGTGCCATGGATGAAC-3′ Primer 69:  (SEQ ID. NO: 155) 5′-AAGTGACCTGCCATTGCGCG*CCAGGTATGTC TACAGCAGAGGGACCCAGC-3′ Primer 70: (SEQ ID. NO: 156) 5′-GGCTTAGAGCACCGCGTCATT*CCAGGTGTCG CTACTGGAAGCGGTGATC-3′ Primer 71: (SEQ ID. NO: 157) 5′-GCGATAGCTAAGGTACGACGGGTC-3′ Primer 72:  (SEQ ID. NO: 158) 5′-GTAGATTCGATCCATGCTCCTCTACTACC-3′ Primer 73: (SEQ ID. NO: 159) 5′-CGTCTTACATGCGCAAGCGG*CCAGGTGATATT GAGTTCGGTAATGCAAGATCTGC-3′ Primer 74: (SEQ ID. NO: 160) 5′-CCATAGAGATGGCAATAGATGAAGAGC-3′ Primer 75:  (SEQ ID. NO: 161) 5′-AGGCGTTCCGCTTCAACGAG*CCAGGTTGTCAGA TTCTGTAGCTTGCTCAGTC-3′ Primer 76: (SEQ ID. NO: 162) 5′-GGTGGTGATCCCAACTTGTTATATCGAAG-3′ Primer 77: (SEQ ID. NO: 163) 5′-TCCGTCTGCGAAGATCTGAGC*CCAGGTTCAATCT ATCRTCTGACAGATCTTGAAGT-3′ Primer 78:  (SEQ ID. NO: 164) 5′-GTGTCACGACGCGCGAATCT*CCAGGAGATCGTGA CCAGTATAATAGCTCAACAC-3′ Primer 79: (SEQ ID. NO: 165) 5′GTTCAGACAATGCAGGGATAACACCAGC-3′ Primer 80: (SEQ ID. NO: 366) 5′-CCCAGAACGATTTGCGGCGT*CCAGGCTTGGTC CTCTCTTAGGAGGCAAGC-3′ Primer 81: (SEQ ID. NO: 167) 5′-AGGATGCTTCGGACTACCTGAG-3′ Primer 82: (SEQ ID. NO: 168) 5′-TGCATTGCCGTCGCAGAGAC*CCAGGCAACGGG CACGAAGCGCATC-3′ Primer 83: (SEQ ID. NO: 369) GCCCTAATGATAAGACAGGCAGTTGTGG Primer 84:  (SEQ ID. NO: 170) 5′-ATGCGCTTGGATTGCCGATG*CCAGGAGCCCTGT TAGTTCTGGATGCTGAACA-3′ SCO 33: (SEQ ID. NO: 171) CTTATAGATTATA[T(FAM)]TGCCCCGTTGAGAGC ACGAAT[BHQ1]  SCO 34: (SEQ ID. NO: 172) CTAAGTAAGCCTATATCGAAT[T(FAM)]CCGTGGC GCGAAGTTATCCA[BHQ1] SCO 35: (SEQ ID. NO: 173) CGTACTGCACTCGCCTACGAC [T(Cal Fluor Red 610) AAGTGACCTGCCATTGCGCG[BHQ2] SCO 36: (SEQ ID. NO: 174) CTTATAAGTTACA[T(Cal Fluor Red 610)]GGC TTAGAGCACCGCGTCATT[BHQ2] SCO 37: (SEQ ID. NO: 175) CTAATTGTAATAC[T(Quasar 670)]CGTCTTACA TGCGCAAGCGG[BHQ2] SCO 38: (SEQ ID. NO: 176) CTAATCGTATGAGATCTATGA[T(Quasar 670)]  AGGCGTTCCGCTTCAACGAG[BHQ2] SCO 39: (SEQ ID. NO: 177) TCATAGACATTTA[T(Cal Fluor Gold 540) TCCGTCTGCGAAGATCTGAGC[BHQ1] SCO 40:  (SEQ ID. NO: 178) TACGAATCTGACCTAGTAAGA [TYCal Fluor Gold 540)]GTGTCACGACGCGCGAATCT[BHQ1] SCO 41: (SEQ ID. NO: 179) TGCCACTAACAGGCCGCTAGA[T(Cal Fluor Gold 540)]CCCAGAACGATTTGCGGCGT[BHQ1] SCO 42:  (SEQ ID. NO: 180) TCGAGCGTGCGCCAGATCCA[T(Quasar 670) TGCATFGCCGTCGCAGAGAC[BHQ2] SCO 43: (SEQ ID. NO: 181)  TCGACTGTGCCTGCGTCCGTA[T(FAM)]ATGCGCTTG GATTGCCGATG[BHQ1] Amplified product 32: GenBank: KU558787.1/Position (start-end): 428-621

(SEQ ID. NO: 182) TTGCTATGGCTGACGGGGAAGAATGGTTTGTACCCAAACCTGAGC ATGTCCTATGTAAACAACAAAGAGAAAGAAGTCCTTGTGCTATGG GGTGTTCATCACCCACCTAACATAGGGAACCAAAGGGCCCTCTAC CATACAGAAAATGCTTATGTCTCTGTAGTGTCTTCACATTATAG CAGAAGATTCACCCC*

ATTCGTGCTCTCAACGGGGC Amplified product 33: GenBank: CY221934.1/Position (start-end): 111-296

(SEQ ID. NO: 183) GGGCACCATGCAGTACCAAACGGAACGATAGTGAAAACAATCACAA ATGACCAAATTGAAGTTACTAATGCTACTGAGTTGGTTCAGAATTC CTCAATAGGTGAAATATGCGACAGTCCTCATCAGATCCTTGATGGA GAGAACTGCACACTAATAGATGCTCTATTGGGAGACCCTCA  GTGTGAT*

TCGATAAGTTCGCGCCACGG  Amplified product 34: GenBank: CY221750.1/Position (start-end): 1291-1501

(SEQ ID. NO: 184) TCAAAGACTAAGTGGTGCCATGGATGAACTCCACAACGAAATACT CGAGCTGGATGAAAAAGTGGATGACCTCAGAGCTGACACTATAAG CTCACAAATAGAACTTGCAGTCTTGCTTTCCAACGAAGGAATAAT AAACAGTGAAGATGAGCATCTATTGGCACTTGAGAGAAAACTAAA GAAAATGCTGGGTCCCTCTGCTCTAGACATA*

CGCGCA ATGGCAGGTCACTT Amplified product 35: GenBank: JF719743.1/Position (start-end): 1816-1950

(SEQ ID. NO: 185)  GGCTTAGAGCACCGCGTCATT*

TGTCGCTACTGGAAG CGGTGATCCGCACAGTGACGACTTTACAGCAATTGCTTACTTA AGGGACGAATTGCTCGCAAAGCATCCGACCTTAGGTTCTGGTA ATGACGAGGCGACCCGTCGTACCTTAGCTATCGC Amplified product 36: GenBank: KX639498.1 z/Position (start-end): 4035-4253

(SEQ ID. NO: 186) GTAGATTCGATCCATGCTCCTCTACTACCATGGTCCAGCCGACTG AGACAAGGGATGATATATAATGCCAATAAAGTAGCTCTGGCACCC CAATGTCTCCCAGTCGACAAAGATATCAGATTCAGAGTrGTATTT GTCAACGGAACATCACTGGGTAGAATCACAATTGCCAAGGTCGCA AAAACTCTTGCAGATCTTGCATTACCGAACTCAATATCA*

CCGCTTGCGCATGTAAGACG Amplified product 37: GenBank: KY369876.1/Position (start-end): 1310-1463

(SEQ ID. NO: 187) CCCATAGAGATGGCAATAGATGAAGAGCCAGAACAATTCGAACA TAGAGCAGACCAAGAACAAGATGGGGAACCTCAATCATCTATAA TCCAATATGCTTGGGCAGAAGGAAACAGAAGCGATGAGCGGACT GAGCAAGGTAGAGAATCTGACAA*

CTCGTTTGAAGC GGAACGCCT Amplified product 38: GenBank: KX894800.1/Position (start-end): 11378-11529

(SEQ ID. NO: 188) GGTGGTGATCCCAACTTGTTATATCGAAGTTTCTATAGAAGAAC TCCTGATTTCCTCACAGAGGCTATAGTTCACTCTGTGTTCATAC TTAGTTATTATACAAACCATGATTTAAAGGATAAACTTCAAGAT CTGTCAGAYGATAGATTGAA*

GCTCAGATCTTCGCAG ACGGA Amplified product 39: GenBank: KY249683.1/Position (start-end): 11465-11577

(SEQ ID. NO: 189) GGTGGTGATCCTAATTTGTTATATCGAAGC TTTTATAGGAGAACTCCAGACTTCCTTACA GAAGCTATAGTACATTCAGTGTTCGTGTTG AGCTATTATACTGGTCACGATCT*

AGATTCGCGCGTCGTGACAC Amplified product 40: GenBank: KJ627391.1/Position (start-end): 3631-3933

(SEQ ID. NO: 190)  TTTCAGACAATGCAGGGATAACACCAGCA ATATCATTGGACCTAATGACTGATGCTGA ACTGGCCAGAGCTGTATCATACATGCCAA CATCTGCAGGGCAGATAAAGCTGATGTTG GAGAACCGCGCAATGGTAAGGAGAAAAGG ATTTGGAATCCTAATAGGGGTCTACGGAA GCTCTGTGATTTACATGGTTCAATTGCCG ATCTTTGGTGTCATAGATACACTTGTTGG ATAATCAAGGCAGCTCCCTCTTGCTCAGA AAAAAACGGGAATTATGCTTGCCTCCTAA GAGAGGACCAAG*

ACGCCGC AAATCGTTCTGGG Amplified product 41: GenBank: KT963081.1/Position (start-end): 18437-18598

(SEQ ID. NO: 191) AGGATGCTTCGGAGTACCTGAGTCCGGGTCTGGTGCAGT TCGCCCGTGCAACAGACACCTACTTCAGTATGGGGAACA AGTTTAGAAACCCCACAGTGGCGCCCACCCACGATGTGA CCACCGACCGTAGCCAGCGACTGATGCTGCGCTTCGTGC CCGTTG*

GTCTCTGCGACGGCAATGCA  Amplified product 42: GenBank: CY221624.1/Position (start-end): 988-1252

(SEQ ID. NO: 192) GCCCTAATGATAAGACAGGCAGTTGTGGTCCAGTATCGTC TAATGGAGCAAATGGAGTAAAAGGATTTTCATTCAAATAC GGCAATGGTGTTTGGATAGGGAGAACTAAAAGCATTAGTT CAAGAAAAGGTTTTGAGATGATTTGGGATCCGAATGGATG GACTGGGACTGACAATAAATTCTCAATAAAGCAAGATATC GTAGGAATAAATGAGTGGTCAGGGTATAGCGGGAGTTTTG TTCAGCATCCAGAACTAACAGGGCT*

CATCGGCAATCCAAGCGCAT

The bold and slanted font of the Primer sequence means the restriction enzyme recognition sequence, and the underline is the complementary sequence of the CCTF produced thereby. the part represented by * is a tag that modified dCTP was inserted into C in the recognition sequence to block the site cleaved by the PspGI restriction enzyme. In SCO, the parentheses mean the position of the nucleotide sequence in which the fluorescent offsetting molecule and the fluorescent reporter are located. The sequence of the CCTF produced from the amplified product is as follows.

CCTF 33: (SEQ ID. NO: 193) 5′-ATTCGTGCTCTCAACGGGGC-3′ CCTF 34: (SEQ ID. NO: 194) 5-TCGATAAGTTCGCGCCACGG-3′ CCTF 35:  (SEQ ID. NO: 195) 5′-CGCGCAATGGCAGGTCACTT-3′ CCTF 36: (SEQ ID. NO: 196) 5′-AATGACGCGGTGCTCTAAGCC-3′ CCTF 37: (SEQ ID. NO: 197) 5′-CCGCTTGCGCATGTAAGACG-3′ CCTF 38: (SEQ ID. NO: 198) 5′-CTCGTTGAAGCGGAACGCCT-3′ CCTF 39: (SEQ ID. NO: 199) 5-GCTCAGATCTTCGCAGACGGA-3′ CCTF 40: (SEQ ID. NO: 200) 5′-AGATTCGCGCGTCGTGACAC-3′ CCTF 41: (SEQ ID. NO: 201) 5′-ACGCCGCAAATCGTTCTGGG-3′ CCTF 42: (SEQ ID. NO: 202) 5-GTCFCTGCGACGGCAATGCA-3′ CCTF 43: (SEQ ID. NO: 203) 5′-CATCGGCAATCCAAGCGCAT-3′

2) PCR Amplification and Determination of SCO Inherent Dissociation Temperature

The following PCR reaction was performed using CFX96 Real-time PCR (Bio-Rad, USA) with 20 μ

of total reaction solution of each of 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 and SCO 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 prepared by adding 0.15 μM, PspGI (NEB, USA) 1U, PCR buffer (1×), MgCl₂ 2.5 mM, dNTP 200 IM, DTT 0.1 mM, RNase Inhibitor 1U, SuperiorScript III (Enzynomics, Korea) 1U Flu A/H1N1, Flu A/H3N2, Flu A/H1N1/2009pdm, the template nucleic acid of the genomic RNA of Flu B, PIV1, PIV3, RSV A, RSV B, hMPV, ADV and MS2 phage 1×10{circumflex over ( )}⁴ copies/rx, respectively.

55° C. 20 mins, 95° C. 10 mins

95° C. 30 secs, 63° C. 1 min (50 cycles).

A reaction was repeatedly performed with a cycle at the reverse transcription reaction temperature of 55° C. for 20 minutes once, and with a cycle at the denaturation temperature of 95° C. for 30 seconds, and an annealing temperature of 63° C. for 1 minute 50 times repeatedly. After the reaction, the reaction mixture was cooled to 50° C. in the same apparatus, held at 50° C. for 30 seconds, and then slowly heated from 50° C. to 95° C. to obtain an inherent dissociation temperature analysis curve. Data analysis was performed with Bio-Rad CFX Manager 1.6.

FIG. 6 shows the results of multiple inherent dissociation temperature measurements for causative organisms of Flu A/H1N1, Flu A/H3N2, Flu A/H1N1/2009pdm, Flu B, PIV1, PIV3, RSV A, RSV B, hMPV, ADV, EC(Ms2 phage). It was confirmed that the peak was observed at the inherent dissociation temperature that each SCO has (Flu A/H1N1: 67.5° C., Flu A/H3N2: 76.5° C., Flu A/H1N1/2009pdm: 86.5° C., Flu B: 83.5° C., PIV1: 66° C., PIV3: 74° C., RSV A: 63.5° C., RSV B: 72° C., hMPV: 86° C., ADV: 85° C.) (a)(b)(c)(d)(e), and no peak of SCO visualizing CCTF was observed when the target sequence was not added in the same composition (f).

Therefore, it was proved that the target nucleic acid sequence can be detected more quickly and simply than the conventional PCR method by analyzing the fluorescence of the SCO using the real-time PCR instrument, simultaneously with the PCR using the marking technique of CCTF.

5. Formation of CCTF and Analysis for the Inherent Dissociation Temperature Peak of CCTF in Multi-Target PCR for Analyzing the Single Nucleotide Polymorphism Genotype of BDNF Gene

CCTF analysis was performed with Real-time PCR instrument for analyzing the genotype of rs6265, single nucleotide polymorphism of BDNF gene.

1) Primer for the Target Sequence of Template DNA, Constructed in the Sequence-Specific Manner

The forward primer used in this example was CTPO and was constructed on the same principle as in Example 1 above. The 5′end of CTPO was composed of 19-20 mers of nucleotide sequences, and was composed of a sequence non-complementary to DNA of the target sequence so as to form CCTF. The restriction enzyme recognition sequence was then located, and from this up to the 3′ end, a sequence complementary to each target site was composed to play a role as a primer. The reverse primer was composed of sequence complementary to the target site to be amplified.

In addition, SCO, which forms a complementary bond with CCTF to be a double-stranded template, was positioned by positioning fluorescent offsetting molecules (BHQ-1 or BHQ-2), and the fluorescent reporter molecular was positioned so as to have a certain distance.

Primer information and target sequence information which is amplified and generated are as follows.

Primer 85: (SEQ ID. NO: 204) 5′-ACGAGGCCTGTCCGCTTACTAG*CCAGGCTG GTCCTCATCCAACAGCTCTTCTATCGC-3′ Primer86: (SEQ ID: NO: 205) 5′-CCGGGTACGCTAAGTCCGCTAT*CCAGGTTCT GGTCCTCATCCAACAGCTCTTCTATCGT-3′ Primer 87: (SEQ. ID. NO: 206) 5′-GACCCATGGGACTCTGGAGAGCGTGAA-3′ Primer 88: (SEQ ID. NO: 207) 5′-GCTCATATGCGGCGCCATTTA*CCAGGGCAG GTTGCTATCAAGGTTACAAGACAG-3′ Primer 89: (SEQ ID. NO: 208) 5-CCGAGCACTTTCTTGCCATGAGCC-3′ SCO 44: (SEQ ID. NO: 209) GTAGCACGCTTCGAATGGC[T(HEX)]ATACGAG GCCTGTCCGCTTACTAG[BHQ1] SCO 45: (SEQ ID. NO: 210) GATACGGAGGTCCGAAGGCAG[T(FAM)]GTTGGT TACCCTAACGCGCCGGA[BHQ1] SCO 46: (SEQ ID. NO: 211) ATTAGTTTAACTATTATATT[T(FAM)]TATGCT CATATGCGGCGCCATTTA[BHQ1] Amplified product 43: GenBank: NT_009237.19/Position (start-end): 27598340-27598451

(SEQ ID. NO: 212) ACGAGGCCTGTCCGCTTACTAG*

CTGGTCCTCA TCCAACAGCTCTTCTATCACGTGTTCGAAAGTGTCAGCCA ATGATGTCAAGCCTCTTGAACCTGCCTTGGGCCCATTCAC GCTCTCCAGAGTCCCATGGGTC Amplified product 44: GenBank: NT_009237.19/Position (start-end): 17598338-7598451

(SEQ ID. NO: 213) CCGGGTACGCTAAGTCCGCTAT*

TTCTGGTCCTCAT CCAACAGCTCTTCTATCACGTGTTCGAAAGTGTCAGCCAATG ATGTCAAGCCTCTTGAACCTGCCTTGGGCCCATTCACGCTCT CCAGAGTCCCATGGGTC Amplified product 45: GenBank: EF450778.1/Position (start-end): 431-681

(SEQ ID. NO: 214)  GCTCATATGCGGCGCCATTTA*

GCAGGTTGCTA TCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACT GGGCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAG GCACTGACTCTCTCTGCCTATTGGTCTATTTTCCCACCCT TAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTT GAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGG GCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGG

The bold and slanted font of the Primer sequence means the restriction enzyme recognition sequence, and the underline is the complementary sequence of the CCTF produced thereby. the part represented by * is a tag that modified dCTP was inserted into C in the recognition sequence to block the site cleaved by the PspGI restriction enzyme. In SCO, the parentheses mean the position of the nucleotide sequence in which the fluorescent offsetting molecule and the fluorescent reporter are located. The sequence of the CCTF produced from the amplified product is as follows.

CCTF 44: (SEQ ID. NO: 215) 5′-CTAGTAAGCGGACAGGCCTCGT-3′ CCTF45: (SEQ ID. NO: 216) 5′-ATAGCGGACTTAGCGTACCCGG-3′ CCTF 46: (SEQ ID. NO: 217) 5′-TAAATGGCGCCGCATATGAG-3′

2) PCR Amplification and Determination of SCO Inherent Dissociation

PCR reaction was performed using the following CFX96 Real-time PCR (Bio-Rad, USA) with 20 μ

of total reaction solution of each of Primer 85, 86, 87, 88, 89 and SCO 44, 45, 46 prepared by adding 0.15 μiM, PspGI (NEB, USA) 5U, PCR buffer (1×), MgCl₂ 2.5 mM, dNTP 200 μiM, h-Taq DNA polymerase (Solgent, Korea) 1.6 U and Flu A/H1N1, Flu A/H3N2, Flu A/H1N1/2009pdm, the template nucleic acids of the genomic RNA of Flu B, PIV1, PIV3, RSV A RSV B, hMPV, ADV and MS2 phage 1×10{circumflex over ( )}⁴ copies/rxn, respectively.

95° C. 15 mins,

95° C. 30 secs, 63° C. 1 min (50 cycles).

A reaction was performed using a cycle at the denaturation temperature of 95° C. for 15 minutes once, and with a cycle at the denaturation temperature of 95° C. for 30 seconds, and an annealing temperature of 63° C. for 1 minute 50 times repeatedly. After the reaction, the reaction mixture was cooled to 50° C. in the same apparatus, held at 50° C. for 30 seconds, and then slowly heated from 50° C. to 95° C. to obtain an inherent dissociation temperature analysis curve. Data analysis was performed with Bio-Rad CFX Manager 1.6.

FIG. 7, (a) shows the results of multiple inherent dissociation temperature measurements for the genotype of mutant type A/A, wild type G/G and heterozygote A/G of rs6265 and IC. It was identified that the peak was observed at the inherent dissociation temperature that each SCO has (A/A: 76.5° C., A/G: 76.5° C.

75° C., G/G 75° C., IC: 66° C.) (a)(b)(c)(d), and no peak of SCO visualizing CCTF was observed when the target sequence was not added in the same composition (e).

Therefore, it was proved that the target nucleic acid sequence can be detected more quickly and simply than the conventional PCR method by analyzing the fluorescence of the SCO using the real-time PCR instrument, simultaneously with the PCR using the marking technique of CCTF.

Example 3. Formation of CCTF and Analysis for Ct Graph of CCTF in Multiple Target PCR

It has been proved in Example 2 that SCO can be used to confirm whether CCTF is generated with a real-time PCR device. The SCO used in the above method is simultaneously formed during the reaction in which the target sequence is generated during the PCR amplification process, and it is possible to identify CCTF generated by real-time fluorescence analysis. Based on this, the present example demonstrated that a standard curve formation is possible when analyzing the formation of CCTF using SCO in the case of PCR with multiple target sequences.

In order to perform this experiment, the causative organisms of sexually transmitted infections (STI), Neisseria. gonorrhea (NG), Mycoplasma. hominis (MH), Ureaplasma. parvum (UP) were selected.

1. Construction of Specific Primer of Target Template DNA

The forward primer used in this example was constructed based on the method described in the detailed description of the invention above as CPTO. The 5′end of the forward primer was composed of a 19-mer or 21-mer nucleotide sequence, and was composed of non-complementary sequences to DNA of each causative organism so as to form CCTF. The restriction enzyme recognition sequence was then consecutively located. After this up to the 3′ end, a sequence complementary to DNA of each causative organism was composed to play a role as a primer. The reverse primer was composed of sequence complementary to the target site of DNA by each causative organism.

In addition, SCO, which forms a dimer with CCTF, was designed to have a double tag, and was separately designed for each causative organism. SCO was designed by positioning quencher (BHQ-1 or BHQ-2) at 3′ end, with reporter molecular (each FAM, HEX, CAL Fluor Red 610) positioned at a certain distance, and its sequence was complementary to CCTF sequence to be analyzed.

Primer information and target sequence information which is amplified and generated are as follows.

Primer 90: (SEQ ID. NO: 218) 5′-CTCATCGCCACGAGCCGGTTAA*

TTGAAACACCGCCCGGAACCC-3′ Primer 91: (SEQ ID. NO: 219) 5′-GCTCCTTATTCGGTTTGACCGGT-3′ Primer 92: (SEQ ID. NO: 220) 5′-GCTCGCAGGTACGGCACCATTCA*

CAGAAGGTA TGATAACAACGGTAGAGC-3′ Primer 93:  (SEQ ID. NO: 221) 5′-CCCCTTTGCACCGTTGAGGGG-3′ Primer 94: (SEQ ID. NO: 222) 5′-AGTCGATTATGTCTGAGGCCGCG*

TTAAAGT AGCATATGATCAAGCTCATTCA-3′ Primer 95: (SEQ ID. NO: 223) 5′-GATCCTGACATATAATCATTATCTCCTTTTATAAA-3′ SCO 47: (SEQ ID. NO: 224) TC[T(HEX)]CATCGCCACGAGCCGGTTAA[BHQ] SCO 48: (SEQ ID. NO: 225) TG[TTCAL Fluor Red 610)]CGCAGGTACGGCACC ATTCA[BHQ2] SCO 49: (SEQ ID. NO: 226) TAG[T(FAM)]CGATTATGTCTGAGGCCGCG[BHQ] Amplified product 46: GenBank: X52364.1/Position (start-end): 375-459

(SEQ ID. NO: 227) CTCATCGCCACGAGCCGGTTAA

TTGAAACACCG CCCGGAACCCGATATAATCCGCCCITCAACATCAGTGAAA ATCTTTTTTTAACCGGTCAAACCGAATAAGGAGC Amplified product 47: GenBank: M31431.1/Position (start-end): 1455-1535

(SEQ ID. NO: 228) GCTCGCAGGTACGGCACCATTCA*

CAGAAGG TATGATAACAACGGTAGAGCTTTATATGATATTAACTT AGCAAAAATGGAAAACCCCTCAACGGTGCAAAGGGG  Amplified product 48: GenBank: AF085733.2/Position (start-end): 416-502

(SEQ ID. NO: 229) AGTCGATTATGTCTGAGGCCGCG*

GTTTCTGTAC ACGATCCAATT[T/c]ACAAATAACATTTACAATTCGTAAA ATTTTTTTATAAAAGGAGATAATGATTATATGTCAGGATC

The bold and slanted font of the Primer sequence means the restriction enzyme recognition sequence, and the underline is the complementary sequence of the CCTF produced thereby. the part represented by * is a tag that modified dCTP was inserted into C in the recognition sequence to block the site cleaved by the PspGI restriction enzyme. In SCO, the parentheses mean the position of the nucleotide sequence in which the fluorescent offsetting molecule and the fluorescent reporter are located. Primer and primer corresponding to NG in SCO is the same as those used in Example 2. The sequence of the CCTF produced from the amplified product is as follows.

CCTF 47: (SEQ ID. NO: 230) 5′-CCTGGTTAACCGGCTCGTGGCGATGAG-3′ CGTF48: (SEQ ID. NO: 231) 5′-CCTGGTGAATGGTGCCGTACCTGCGAGC-3′ CCTF 49: (SEQ ID. NO: 232)  5′-CCTGGCGCGGCCTCAGACATAATCGACT-3′

2. PCR Amplification and Determination of SCO Inherent Dissociation

PCR reaction was performed using the following CFX96 Real-time PCR (Bio-Rad USA) with 20 μ

of total reaction solution obtained by adding three kinds of the specific forward primers and three kinds of reverse primers of each target sequence, as mentioned in the above primer design, and three kinds of SCO to be 0.15 μM, respectively, and adding PspGI (NEB, USA) 2 U, PCR buffer (1×), MgCl₂ 2.5 mM, dNTP 200 μM, h-Taq DNA polymerase (Solgent, Korea) 1.6 U, and contained the template DNA diluted by 10-folds with 100 pg/μl genomic DNA proven by the conventional quantitation method for each causative organism.

95° C. 15 mins,

95° C. 30 secs, 63° C. min (50 cycles).

A reaction was repeatedly performed with a cycle at the denaturation temperature of 95° C. for 15 minutes once, and with a cycle at the denaturation temperature of 95° C. for 30 seconds, and an annealing temperature of 63° C. for 1 minute 50 times. In addition, fluorescence signals were collected at the annealing stage, and the data analysis was performed with Bio-Rad CFX Manager 1.6. Cycle threshold (Ct) was started with an algebraic amplifier using a known number of DNA concentrations to create a standard curve for the strain.

As shown in (a) of FIG. 8, the expected fluorescence amplification curves of SCO could be observed with each of different graphs depending on the concentration of the template. Also, any peak was observed when the template DNA was not added (b). As the results showing fluorescence amplification curves and standards of SCO represented by the experimental condition of Polymerase Chain Reaction of NG (solid line), MG (dotted line), and UP (circle), dilutions for genomic DNA of each causative organism diluted by 10-folds starting from the concentration of 100 μg, graph (a) indicates the fluorescence amplification curve drawn when the three target sequences are present at the same time by the concentration, graph (b) is the negative result drawn when all three target sequences are not included. When the graph corresponding to NG in graph (a) is represented by the single fluorescence amplification curve and thus the standard curve, it can be represented by (c) and (d), respectively. The graph corresponding to MG can be expressed by (e) and (f), respectively, and the curve corresponding to UP can be represented by (g) and (h), respectively.

Regression coefficient (r²) in the linear regression analysis of the standard curve was represented by NG 0.9982, MG 0.999, UP 0.9992, respectively. The slope of the regression plot was NG −3.85, MG −3.89, and UP −3.66, respectively. It could be identified that the respective amplification efficiency (E=10^([−1/slope])−1) was 81.8% for NG, 80.7% for MG and 87.6% for UP, respectively, and thus, they were listed in the proper range of between 80 and 120%.

From this Example, when reading the different CCTFs by each of causal organisms using the real-time PCR instrument, it was demonstrated that the relative amount of CCTF to be generated by measuring a degree of the real-time fluorescence of SCO is grasped, and by using this, the Ct value is confirmed, and therefore, the identifying of the target sequence is possible. 

What is claimed is:
 1. A method for forming and identifying a tag used in classifying and analyzing kinds of the target sequences amplified in the Polymerase Chain Reaction, which comprises: a) hybridizing a target sequence with a primer comprising a template of a tag for generating the tag, which is a cleaved complementary tag fragment; b) generating the complementary tag fragment cleaved from the primer by an activity of a restriction enzyme when the amplification procedure is proceeded by the hybridization of a) step and releasing and introducing it into a reaction solution; and c) identifying the generated cleaved complementary tag fragment through an analyzer to confirm the presence of the target nucleic acid sequence wherein said primer of a) step comprises a random nucleic acid sequence noncomplementary to a target sequence and has a structure sequentially comprising a restriction enzyme recognition sequence and a nucleic acid sequence complementary to the target sequence.
 2. The method according to claim 1, characterized in that the restriction enzyme recognition sequence is the recognition sequence for the restriction enzyme selected from the group consisting of Pho I, PspGI, BstNI, TfiI, ApeKI, TspMI, BstBI, BstEII, BstNI, BstUI, BssKI, BstYI, TaqI, MwoI, TseI, Tsp45I, Tsp509I, TspRI, Tth111I, Nb.BsmI, Nb.BsrDI, NLBspQI, Nt.BstNBI restriction enzymes and Nick restriction enzyme.
 3. The method according to claim 1, characterized in that the modified dNTP is inserted in a region of the primer cleaved by a restriction enzyme in order to prevent cleaved by-products other than the cleaved complementary tag fragment from participating in the reaction.
 4. The method according to claim 3, characterized in that the modified dNTP inserted in the cleaved region comprises Phosphorothioated dNTP, dNTP comprising 7-Deazapurine, or 2′-O-methyl nucleotide(2′-OMeN) in DNA template.
 5. The method according to claim 1, characterized in that the said method analyzes the mass of the cleaved complementary tag fragment to identify the cleaved complementary tag fragment.
 6. The method according to claim 5, characterized in that the instrument used for the mass spectrometry is a matrix-assisted laser desorption-ionization-time-of-flight mass spectrometer (MALDI-TOF MS), a Liquid Chromatography Mass Spectrometer, or a High Performance Liquid Chromatography Mass Spectrometer.
 7. The method according to claim 6, characterized in that the mass per unit electric charge (m/z) of the cleaved tag fragment used for mass spectrometry is present in the range of from greater than 0 to 10000 Da or less.
 8. The method according to claim 7, characterized in that DNA polymerase that the function of adenine addition elongation effect (A tailing) at the 3′ end, being an inherent property of the polymerase is inhibited, is used in order to preserve the mass of a cleaved complementary tag fragment used in mass analysis during the amplification process.
 9. The method according to claim 1, characterized in that the fluorescence signal is analyzed by using the oligonucleotide that is tagged by fluorescence and Quencher and has the complementary sequence of the cleaved complementary tag fragment, as the identification method of the cleaved complementary tag fragment.
 10. The method according to claim 9, characterized in that the said method analyzes the dissociation temperature and melting peak by varying the inherent dissociation temperature at which the double strand of the oligonucleotide and the cleaved complementary tag fragment are dissociated into a single strand, and identifies the cleaved complementary tag fragment to confirm the presence of the target sequence.
 11. The method according to claim 9, characterized in that the said method is made to have different dissociation temperatures to simultaneously analyze two or more kinds of targets through a melting peak analysis in the case of that two or more targets are detected.
 12. The method according to claim 9, characterized in that the oligonucleotide is from 5 or more to 50 or less in length.
 13. The method according to claim 9, characterized in that the nucleotide at the 3′end of the oligonucleotide is blocked in order to prevent elongation of the base sequence from the oligonucleotide.
 14. The method according to claim 13, characterized in that the said method attaches Spacer C3, Phosphat, ddC, or Inverted END to the nucleotide at the 3′end of the oligonucleotide in order to prevent elongation of the base sequence from the oligonucleotide.
 15. The method according to claim 13, characterized in that the said method attaches the quencher to the nucleotide at the 3′end of the oligonucleotide is blocked in order to prevent elongation of the base sequence from the oligonucleotide.
 16. The method according claim 1, characterized in that the method identifies a causative organism of the sexually transmitted disease, the causative organism of gastrointestinal tract disease, a Human Papilloma virus, a causative organism of the respiratory disease, or a gene type of a single nucleotide polymorphism (SNP).
 17. The method according to claim 9, characterized in that the method identifies the complementary tag fragment cleaved by analyzing the cycle threshold (Ct) value of the fluorescence signal of the oligonucleotide. 